In the 1820's, the town of St. Marks, Florida, was considered an important port of entry. The town served as a port for the prosperous planting region of Middle Florida and some counties of South Georgia. Growers hauled their agricultural products down to the port town in wagons by way of an early road that connected the then territorial capital of Tallahassee to the town of St. Marks. Later, this road would be widened and improved upon by the Tallahassee Railroad Company and would become the state's first railroad.

Once the agricultural products reached the new port town, they were loaded aboard boats for shipment to New Orleans and/or St. Augustine. There were, however, problems in navigating both the Apalachee Bay and the St. Marks River. In many places, both bay and river were shallow, and it was common for boats to run aground and/or be stuck in the muddy shallows. In 1828, Florida's Territorial Governor William P. DuVal wrote a letter to Joseph M. White, a territorial delegate in which he stressed a great need for a lighthouse at the St. Marks location. White, in turn, wrote a letter to New Hampshire Senator Levi Woodbury, who chaired the Senate Committee on Commerce, reiterating the importance of establishing a light at St. Marks. Eleven days later, the committee issued a report, which recognized the town of St. Marks as an official port of entry and recommended the building of a lighthouse in the area. On May 23, 1828, the U.S. House of Representatives passed an act, which authorized the construction of a lighthouse at St. Marks and appropriated $6,000 for its construction.

After Robert Mitchell, the Collector of Customs at Pensacola, and a site chosen for the lighthouse completed a survey of the St. Marks area, it was discovered that the initial construction sum of $6,000 would be insufficient. The appropriation was increased to $14,000, and by mid 1829, a contract was signed with Winslow Lewis of Boston for the construction of a tower in the St. Marks area for $11,765. The finished product was place in service in March of 1830. However, a dispute arose over the construction method. The tower was built of brick and stone, whereas the contract specified brick or stone. The government declared the contractors in 'breach of contract' and ordered it be rebuilt according to specifications.  Calvin Knowlton was brought in to rebuild the tower. He oversaw its completion and in 1831 and the Collector of Customs for St. Marks, Mr. Jesse H.  Williams, satisfied that the light was built according to the contract, accepted the work. That same year saw the tower's whale oil lamps lit by Samuel Crosby, who had been appointed the first Keeper of the St. Marks Lighthouse the previous year.

Samuel Crosby was still keeper of the St. Marks Lighthouse in 1835, when the Second Seminole Indian War broke out, and soon learned of the Indian attacks on both the Cape Florida and Mosquito Inlet lighthouses. Fearing for the safety of his family, Crosby wrote authorities and requested that a small detachment of troops be quartered near his lighthouse in order to protect both it and his family against hostile attack. His request was refused. Crosby, still not satisfied with his situation, again wrote authorities and this time requested that a small boat be provided, which he could use to evacuate his family in the event of an emergency. This request was also refused. Fortunately, the Seminole Indians chose not to attack the St. Marks Lighthouse and Crosby continued in his duties as head keeper for another four years.

In 1842, erosion threatened the lighthouse and Winslow Lewis was again called in. He was given a contract to move the tower to a safer location. Lewis's contractors dismantled and removed the lantern and illuminating apparatus, then tore down the original 1831 brick tower. Another site was selected farther inland, away from the water, and a new tower was constructed, then the original lantern and illuminating apparatus were reinstalled.

The new tower survived the destructive hurricanes of the 1840's and 1850's, including the disastrous hurricane of September 1843, which destroyed most of the town of Port Leon and caused major damage to the town of St. Marks. By the 1860's, however, a new threat to the lighthouse arose: The Civil War. In 1865, Confederate troops were stationed near the lighthouse to defend the area against a Union attack. The tower's lighting apparatus had been removed earlier in order to prevent the lighthouse from aiding ships of the Union blockade, which were patrolling the Apalachee Bay. In March of that year, a Federal fleet of 16 ships appeared off the coast and began to shell the vicinity of the lighthouse in preparation for landing a force.

After the end of the conflict, the tower was repaired between September and December of 1866. The lighthouse received a new fourth-order Fresnel lens, which was first lit by Keeper David Kennedy on January 8, 1867 (Lens later removed in Nov. 2014 for preservation). In 1883, the tower was extended an additional ten feet, raising its focal plane. During this subsequent rebuild, the tower was heightened to its present focal plane of 82 feet above sea level.

Charles Fine served as keeper from 1892 to 1904, when he was succeeded by his wife, Sarah. One of the Fine's daughters was born, and raised at the lighthouse and eventually married J.Y. Gresham, a keeper at the lighthouse. Gresham would serve more years at the lighthouse than any other keeper. The Gresham children were raised in relative isolation at the lighthouse, and a private schoolteacher lived with the family to provide a formal education. One teacher, E.W. Roberts from Mississippi, became enamored with one of the Gresham daughters and eventually married her when she turned twenty-three. Another of the daughters (Eula) became interested in a fisherman (Jesse Bishop) from St. Marks, who frequented the waters near the lighthouse. Knowing her father would not approve of the relationship, the daughter met the fisherman in secret and eventually eloped with him. Their marriage ended sadly in divorce, just as keeper Gresham predicted it would.

During the Gresham's service, the area around the lighthouse was incorporated into the St. Marks National Wildlife Refuge. The refuge now encompasses more than 83,000 acres, which serve as wintering habitat for migratory birds. The Gresham's continued to serve at the lighthouse after the Coast Guard assumed responsibility for the nation's lighthouses in 1939 until his retirement in 1949. Keeper Gresham's son, Anton, a member of the US Coast Guard, then served as Officer-In-Charge for two years.

The Lighthouse was automated in 1960 and remained an active aid to navigation for vessels on the Apalachee Bay. Although the Fresnel lens remained in the tower, it was deactivated and a modern, solar powered beacon was placed outside the lantern room during a renovation in 2001.

In October 2013, ownership of the St. Marks Light was officially transferred from the U.S. Coast Guard to U.S. Fish & Wildlife service, St. Marks NWR. The Fresnel lens was removed during renovation of the lantern in 2014, and is now on display in the refuge Visitor Center. During this renovation, the Coast Guard removed their beacon (2016) and the St. Marks Lighthouse went dark for the first time since the Civil War.

Between November 2017 and April 2018, a major renovation of the keeper's dwelling and lighthouse tower was undertaken. A replica 4th order Fresnel lens was installed in the tower in 2019, and is lit seasonally as a Private Aid to Navigation.