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.