Vitamin D is essential for maintenance of skeletal integrity and calcium homeostasis in vertebrates, and plays extraskeletal roles in cardiovascular, infectious, and reproductive diseases. The vitamin D requirements and predominant metabolites of radiated tortoises (Astrochelys radiata) are currently unknown. LC/MS-MS technology was used to evaluate vitamin D metabolites including 25-hydroxycholecalciferol (25(OH)D3), 25-hydroxyergocalciferol (25(OH)D2), and 24,25-hydroxycholecalciferol (24,25(OH)2D3) in seventeen managed subadult to adult radiated tortoises. Tortoises were housed outdoors in a humid subtropical environment during the summer and indoors under artificial humid subtropical conditions in winter. In peak summer, both serum and plasma were evaluated. There was no significant difference between plasma and serum for any of the three metabolites. 25(OH)D3 was the primary plasma vitamin D metabolite at 9.36 ng/mL (SD +/- 2.43) in summer compared to 25(OH)D2 at 1.08 ng/mL (SD +/- 0.59) and 24,25(OH)2D3 at 0.42 ng/mL (SD +/- 0.23). At the peak of winter, with no artificial UVB supplementation or dietary changes, both 25(OH)D3 and 25(OH)D2 were significantly decreased compared to the summer values (P = <0.0001 and P = 0.001, respectively), and 25(OH)D3 remaining the predominant metabolite. In this population, these results indicate that vitamin D is primarily acquired through cutaneous synthesis. This species is also unable to sustain summer vitamin D levels without winter supplemental UVB lighting or dietary sources. Further studies are needed to determine optimal vitamin D levels and to create recommendations for artificial UVB and/or oral vitamin D supplementation in managed tortoises housed indoors.