We investigated the ecology, distribution, and density of Florida Box Turtle (Terrapene carolina bauri Taylor, 1895) populations in the Ten Thousand Islands (TTI), an estuarine mangrove ecosystem in southwestern Florida. The distribution and ecology of Box Turtles in this region and this habitat type have not been previously investigated. The study area encompassed 18 islands and included five natural islands, 13 man-made shell islands, and adjacent mangrove environments. Two hundred and twenty-nine live Box Turtles and 95 Box Turtle shells were detected a total of 409 times on seven of the 18 islands. The seven islands where Box Turtles were detected ranged in size from 7.3 to 31.0 ha and were ancient shell work sites, apparently constructed by the Calusa or other Prehistoric Indians approximately 1,900 to 900 years before present (ybp). Box Turtles were not detected on natural islands. We detected Box Turtles primarily in subtropical hardwood hammock forests, but we detected 10% of turtles in mangrove or mangrove ecotones. Males were larger than females across all sites. We detected a significant difference in body size between living and dead adults. We estimated population size on four shell work islands to range from 43 (SE = 3.5) turtles to 270 (SE = 244) turtles and estimated densities ranging from 2.7–12.2 turtles/ha. Box Turtles on the shell work islands of the TTI are a unique example of populations living on ancient, manmade islands. However, known populations are small and isolated and may be susceptible to increased human recreational use, mechanized vegetation management, or predation by raccoons.