Objective To investigate the effects of Roux-en-Y gastric bypass (RYGB) on hepatic and peripheral insulin sensitivity in rats with type 2 diabetes and their possible mechanisms. Methods 5-6 week old SPF male Sprague-Dawley rats were divided into 2 groups:diabetic RYGB group (n=21) and diabetic RYGB sham group (n=7). The hyperinsulinemic-euglycemic clamp with tracer infusion was completed at 2, 4, and 8 weeks postoperatively to assess the insulin sensitivity. The lipid content in liver and muscle tissue was examined. Results Postoperatively, the diabetic RYGB group had significant decreases in weight, fat mass, and RYGB had a profound effect on the plasma lipid profile. Two weeks after surgery, the hepatic insulin sensitivity index was significantly improved and the hepatic triglyceride was decreased in the RYGB group (P<0.05). The significant increased insulin sensitivity was not detected until four weeks after RYGB surgery, the M value was significantly increased and the TG content in the muscle tissue was significantly decreased (P<0.05). Conclusions The increased insulin sensitivity after RYGB occurs earlier in the liver than in the muscle and both may contribute to the long-term remission of type 2 diabetes. Reduced lipid content in hepatocytes and skeletal muscle cells after RYGB may contribute to the improved insulin sensitivity in these cells.