Vitamin D supplementation may pose a significant health risk in species where levels of deficiency, sufficiency, and toxicity have not been clearly established, and species-specific research on vitamin D supplementation should be performed. This study documented the effect of vitamin D supplementation on serum vitamin D metabolites and other analytes of Ca homeostasis in Asian elephants (Elephas maximus). Six adult Asian elephants received PO supplementation with cholecalciferol at 300 IU/kg of body weight (BW) once a week for 24 wk. Serum was analyzed every 4 wk for 25-hydroxyvitamin D2/D3 [25(OH)D]; 24,25-dihydroxyvitamin D2/D3 [24,25(OH)2D]; 1,25-dihydroxyvitamin D [1,25(OH)2D]; parathyroid hormone (PTH); total Ca; ionized Ca (iCa); P; and Mg. After the supplement was discontinued, serum 25(OH)D2/D3 was measured every 4 wk until levels returned to baseline. At the start of the study, the average serum 25(OH)D3 was nondetectable (<1.5 ng/ml). With cholecalciferol supplementation, 25(OH)D3 increased at an average rate of 2.26 ng/ml per month and reached an average concentration of 12.9 ± 3.46 ng/ml at 24 wk. Both 24,25(OH)2D3 and 1,25(OH)2D increased over time with supplementation from an average of <1.5 to 12.9 ng/ml and from 9.67 to 36.4 pg/ml, respectively. PTH, iCa, Ca, P, and Mg remained within reported normal ranges throughout supplementation. After the supplement was discontinued, serum 25(OH)D3 demonstrated a slow decline to baseline, taking an average of 48 wk. Elephants demonstrated significant individual variation in response to supplementation and subsequent return to baseline. Supplementation of Asian elephants with a weekly dose of 300 IU/kg BW cholecalciferol for 24 wk appears to be effective and safe. Additional clinical studies would be necessary to investigate the safety of other routes of administration, dosages, and duration of vitamin D supplementation, as well as associated health benefits.