The adipocyte derived hormone leptin has been shown to acutely increase fatty acid (FA) oxidation and decrease esterification in resting rodent skeletal muscle. However, the effects of leptin on human skeletal muscle FA metabolism are completely unknown. In these studies we have utilized an isolated human skeletal muscle preparation combined with the pulse-chase technique to measure FA metabolism with and without leptin in lean and obese human skeletal muscle. Under basal conditions (in the absence of leptin) muscle from the obese demonstrated significantly elevated levels of total FA uptake (+72%, p=0.038) and enhanced rates of FA esterification into triacylglycerol (+102%, p=0.042) when compared to lean subjects. In the presence of leptin, lean muscle had elevated rates of endogenous (+103%, p=0.01) and exogenous (+150%, p=0.03) palmitate oxidation. When examining the esterification to exogenous oxidation ratio, leptin reduced this ratio (-47%, p=0.032) demonstrating the increased partitioning of FA towards oxidation and away from storage. Contrary to these findings in lean muscle, leptin had no effect on FA metabolism in skeletal muscle of the obese. This study provides the first evidence that leptin increases FA oxidation in skeletal muscle of lean, but not obese humans, thus demonstrating the development of leptin resistance in obese human skeletal muscle.