The effect of conjugated linoleic acid (CLA), a popular weight-loss supplement, on insulin sensitivity in humans is controversial and has notbeen extensively studied. To date no studies have examined the effects of CLA supplementation on human skeletal muscle metabolism or lipid content. It is also unknown whether CLA accumulates in human skeletal muscle with supplementation. In the present study, 9 overweight, non-diabetic individuals received 4 g/d of mixed CLA isomers in the form of 1 g supplements, for 12 weeks. CLA isomers significantly increased in both plasma and skeletal muscle following supplementation. Skeletal muscle ceramide content was also significantly increased, although there was no consistent change in muscle diacylglycerol or triacylglycerol content. Insulin sensitivity was significantly decreased (p ≤ 0.05) following CLA supplementation, with a significant increase in glucose and insulin area under the curve during an oral glucose tolerance test (p ≤ 0.005). The protein content of PPARα, for which CLA is a potent ligand, was unchanged following supplementation. Accordingly, there was no change in markers of muscle oxidative capacity. In conclusion, mixed-isomer CLA supplementation in overweight non-diabetic humans results in the accumulation of CLA and ceramide in skeletal muscle, and decreases insulin sensitivity.