Role of opioid mu-receptor that has been showed to involve in the insulin-dependent diabetic rats in the induction of insulin resistance remains unclear. The present study is performed to clarify this point. The wild-type mice or opioid mu-receptor knockout mice were employed to induce insulin resistance by feeding with fructose-rich chow or by the repeated intraperitoneal injections of long-acting form of human insulin at 0.5 IU/kg three times daily. The basal plasma glucose concentration was not markedly changed in fructose-fed mice regardless the presence of opioid mu-receptor or not. However, the plasma glucose lowering activity of tolbutamide (10.0 mg/kg) disappeared rapidly in opioid mu-receptor knockout mice receiving fructose-rich chow as compared to that in wild type group. In opioid mu-receptor knockout mice, the elevation of plasma glucose concentration and the loss of plasma glucose lowering activity of tolbutamide were observed at 15 days after insulin injection. However, similar change was obtained at 21 days later of insulin injection in wild-type mice, showing that decrease of insulin action was more markedly in opioid mu-receptor knockout mice. Our results indicated that opioid mu-receptor is related to the delay of insulin resistance induced by fructose-fed method or insulin repeated injection.