Uncoupling protein-3 (UCP3) is a mitochondrial inner-membrane protein highly expressed in skeletal muscle. While UCP3s function is still unknown, it has been hypothesized to act as a fatty acid (FA) anion exporter, protecting mitochondria against lipid peroxidation, and/or facilitating FA oxidation. The aim of this study was to determine the effects of long-term feeding of a 45% fat diet on whole body indicators of muscle metabolism in congenic C57BL/6 mice that were either lacking UCP3 (Ucp3 -/-) or had a transgenically-induced ~2-fold increase in UCP3 levels (UCP3tg). Mice were fed the high fat (HF) diet for a period of either 4 or 8 months immediately following weaning. After long-term HF feeding, UCP3tg mice weighed an average of 15% less than wild type mice (p<0.05) and were 20% less metabolically efficient than both wild type and Ucp3 -/- mice (p<0.01). Additionally, wild type mice had 21% lower, while UCP3tg mice had 36% lower levels of adiposity compared to Ucp3 -/- mice (p<0.05, p<0.001, respectively), indicating a protective effect of UCP3 against fat gain. No differences in whole body oxygen consumption were detected following long-term HF feeding. Glucose and insulin tolerance tests revealed that both the UCP3tg and Ucp3 -/- mice were more glucose tolerant and insulin sensitive compared to wild type mice after short-term HF feeding, but this protection was not maintained in the long-term. Findings indicate that UCP3 is involved in protection from fat gain induced by long-term HF feeding, but not in protection from insulin resistance.