Feline vitamin D status is based on dietary consumption but metabolism of this essential nutrient and the efficacy of supplementation forms are poorly described in cats. The aim of this study was to further elucidate the metabolites of vitamin D in cats and to compare the effectiveness of vitamin D and 25(OH)D for increasing feline vitamin D status. Eight adult male castrated domestic shorthair cats received vitamin D or 25(OH)D in a single crossover design. Vitamin D was dosed daily in a molar equivalent dosage to vitamin D ingested in the diet while 25(OH)D was provided at a daily dose of 20% molar equivalent intake of dietary vitamin D based on its expected higher potency. Plasma concentrations of 25-hydroxyvitamin D epimers were evaluated at baseline then every 2 weeks for a total of 10 weeks. Analysis of multiple vitamin D metabolite concentrations was completed at the end of each supplementation period, followed by a washout period preceding the second phase of the crossover trial. Results showed that supplementation with 25(OH)D more effectively and rapidly raised circulating 25(OH)D levels in cat plasma compared to vitamin D. Formation of C-3 epimers of 25(OH)D, 25(OH)D, and 24,25(OH)D, but not 24,25(OH)D, were observed in feline plasma. The abundant concentrations of epimeric forms of vitamin D metabolites found in circulation suggest that these metabolites should be considered during vitamin D analyses in cats. Further studies using 25(OH)D and vitamin D forms are needed to conclude safety and efficacy of these vitamers for supplementation in this species.