Increasing evidence supports a negative role of glycogen synthase kinase-3 (GSK-3) in the regulation of skeletal muscle glucose transport activity. We have shown previously that acute treatment of insulin-resistant rodents with selective GSK-3 inhibitors improves whole-body insulin sensitivity and insulin action on muscle glucose transport. In the present investigation, we assessed the effects of chronic treatment of insulin-resistant, pre-diabetic obese Zucker (fa/fa) rats with a highly selective GSK-3 inhibitor (CT118637) on glucose tolerance, whole-body insulin sensitivity, plasma lipids, skeletal muscle insulin signaling, and in vitro skeletal muscle glucose transport activity. Obese Zucker rats were treated by gavage with either vehicle or CT118637 (30 mg/kg body wt) twice per day for 10 days, and studied 15-18 hours after the last treatment. Fasting plasma insulin and free fatty acid levels were reduced by 14% and 23% (p<0.05), respectively, in GSK-3 inhibitor-treated animals compared to vehicle-treated controls. The glucose response during an oral glucose tolerance test was reduced by 18% (P<0.05) and whole-body insulin sensitivity was increased by 28% (P<0.05) following chronic GSK-3 inhibition. In vivo IRS-1 tyrosine phosphorylation (50%) and IRS-1 associated phosphatidylinositol-3[[rad]] kinase (79%) relative to fasting plasma insulin were significantly elevated (P<0.05) in plantaris muscles of GSK-3 inhibitor-treated animals. Whereas basal glucose transport in isolated soleus and epitrochlearis muscles was unaffected by chronic GSK-3 treatments, insulin stimulation of glucose transport above basal was significantly enhanced (32-60%, P<0.05). In summary, chronic treatment of insulin-resistant, pre-diabetic obese Zucker rats with a specific GSK-3 inhibitor enhances oral glucose tolerance and whole-body insulin sensitivity, and is associated with an amelioration of dyslipidemia and an improvement in IRS-1-dependent insulin signaling in skeletal muscle. These results provide further evidence that selective targeting of GSK-3 in muscle may be an effective intervention for the treatment of obesity-associated insulin resistance.