Disorders of calcium homeostasis have been reported with some frequency in two-toed sloths, yet little investigation has been performed on vitamin D and mineral metabolism in these species. This study evaluates biomarkers involved in vitamin D and calcium metabolism in a group of nine managed Hoffmann's two-toed sloths (Choloepus hoffmanni), correlated with diet analysis. Serum was analyzed for both vitamin D₂ and D₃ metabolites, minerals (calcium [Ca], phosphorus [P], and magnesium [Mg]), parathyroid hormone (PTH), and ionized calcium (iCa). The diet was analyzed for proximate nutrients, minerals and vitamins D₂ and D₃, and feed intake was estimated. Average values reported for D₃ metabolites, including 25(OH)D₃, 1,25(OH)₂D₃, and 24,25(OH)₂D₃, were 25.1 ng/ml, 52.23 pg/ml, and 7.65 ng/ml, respectively. Average PTH was 0.22 pmol/L while average iCa was 1.46 mmol/L. Values reported for Ca, P, Mg were within expected ranges. Dietary concentration of D₃ was 6.7 ng/g and the average daily intake per sloth was 113.7 IU/kg body weight. No detectable levels of vitamin D₂ metabolites 25(OH)D₂, 1,25(OH)₂D₂, or 24,25(OH)₂D₂ were found in the diet or serum. These data will serve as a starting point for future investigations into the vitamin D metabolism and calcium homeostasis of two-toed sloths to improve the health in managed settings.