Uncoupling protein (UCP)-2 is a member of the mitochondrial inner membrane carriers that uncouple proton entry in the mitochondrial matrix from ATP synthesis. The −866G/A polymorphism in the UCP2 gene, which enhances its transcriptional activity, was associated with enhanced risk for type 2 diabetes in obese subjects. We addressed the question of whether the −866G/A polymorphism contributes to variation in insulin sensitivity by genotyping 181 nondiabetic offspring of type 2 diabetic patients. Insulin sensitivity, assessed by the hyperinsulinemic-euglycemic clamp, was reduced in −866A/A carriers compared with −866A/G or −866G/G carriers (P = 0.01). To directly investigate the correlation between UCP2 expression and insulin resistance, UCP2 mRNA levels were measured by real-time RT-PCR in subcutaneous fat obtained from 100 obese subjects who underwent laparoscopic adjustable gastric banding. UCP2 mRNA expression was significantly correlated with insulin resistance as assessed by the homeostasis model assessment index (r = 0.27, P = 0.007). We examined the association of the −866A/A genotype in a case-control study including 483 type 2 diabetic subjects and 565 control subjects. The −866A/A genotype was associated with diabetes in women (odds ratio 1.84, 95% CI 1.03-3.28; P = 0.037), but not in men. These results indicate that the −866A/A genotype of the UCP2 gene may contribute to diabetes susceptibility by affecting insulin sensitivity.