Puerarin, a major active isoflavone extracted from the root of Pueraria lobate, significantly increases plasma β-endorphin and insulin levels and improves impaired insulin signaling in diabetic animals. However, the target tissues and underlying mechanisms in and through which puerarin functions to ameliorating insulin resistance remains largely unclear. In this study, we showed that puerarin enhanced μ-opioid receptor expression and phosphorylation, and increased insulin-stimulated glucose transporter 4 translocation to the plasma membrane in the skeletal muscle of diabetic rats, which were recaptured by a direct application of puerarin in the palmitate-induced insulin-resistant L6 myotubes. Naloxone, an antagonist of μ-opioid receptor, blocked these functions of puerarin. No β-endorphin was detected either in the muscle of diabetic rats or in the palmitate-induced insulin-resistant L6 cells. Furthermore, we presented the evidence to show the interaction between μ-opioid receptor and insulin receptor substrate 1 in the muscle tissues and cells. These results suggested that puerarin improved insulin sensitivity in the skeletal muscle at least in part by its local effects involving μ-opioid receptor function.