Serum levels of alpha-tocopherol (vitamin E) and 25-hydroxyvitamin D (vitamin D) were measured in 25 wild Florida manatees (Trichechus manatus latirostris) from Crystal River National Wildlife Refuge, Citrus County, Florida, US. The samples were collected during the 2017 and 2018 winter health assessments. Reference intervals for alpha-tocopherol were 0.15–3.2 µg/L with mean value of 0.76 µg/mL and for 25-hydroxyvitamin D were 4.3–23 nmol/L with a mean of 13.88 nmol/L. Four, long-term captive manatees and nine manatees in rehabilitation were available for comparisons. Alpha-tocopherol levels were within the established range for free-ranging manatees. Mean 25-hydroxyvitamin D levels in long-term captive and manatees in rehabilitation fell well outside the reference interval for free-ranging manatees, being six- to sevenfold higher. The absence of significant differences in alpha-tocopherol between wild and captive manatees may not have any significant direct clinical implications but may warrant an investigation into other antioxidant mechanisms in manatees. The elevated 25-hydroxyvitamin D levels seen in captivity were unexpected and, in a few individuals, may pose a health challenge. Calcium is known to be elevated in captive manatees as well; a close examination of a potential hypervitaminosis D and hypercalcemia may be warranted.