Nutrition most certainly affects health and may play a role in the etiology of growth and reproductive problems in captive cheetah (Acinonyx jubatus) populations. The objective of our research was to examine nutritional differences between two dietary regimens and quantify their physiologic effects on cheetahs held in captivity. Twelve cheetahs were randomly assigned to either a commercial diet (COM) or a supplemented meat diet (SMD) group. These cats were physically examined and had blood samples taken three times over the course of a year. Representative samples of COM and four separate components of the SMD treatment were analyzed over the same time frame for proximate nutrient composition, digestibility, and concentrations of taurine, fat-soluble vitamins, and selected minerals. Concentrations of fat, vitamins A and E, Se, Fe, Cu, Na, and Mn were significantly higher in COM compared with those in SMD samples, with the exception of fat content in turkey. Mg content was lower in COM than in SMD; other nutrients did not differ. Mean concentrations of vitamins A and E in COM were markedly higher than in SMD samples (408,140 vs. 29,696 IU/kg dry matter [DM] and 431 vs. 48 IU/kg DM, respectively) and varied dramatically between sampling periods. Percent crude protein and protein-to-fat ratios were high for SMD compared with either whole prey–based or commercial food preparations. Blood urea nitrogen and serum creatinine levels were above normal reference means for domestic cats. Plasma concentrations of vitamins A, D, and E were significantly higher in COM-fed than in SMD-fed cheetahs. Both plasma retinol and tocopherol levels were almost three times higher in COM-fed cats (1.26 ± 0.06 vs. 0.53 ± 0.03 μg/ml and 17.5 ± 0.7 vs. 6.4 ± 0.02 μg/ml, respectively) and exceeded the normal ranges expected for domestic felids. Significant differences between male and female cheetahs were found for plasma concentrations of vitamin E, Se, and Fe after allowing for effects of diet and time of collection. Excess fat-soluble dietary vitamins can result in direct toxicities as well as nutrient antagonisms and may be linked to reproductive and health issues in captive cheetahs. The high protein levels found in SMD may be linked to chronic renal disease, which was detected in some of these cheetahs.