Background: L-carnitine is known to play a beneficial role in diabetes; however, there are no studies on the effect of L-carnitine on glucose transporter 4 (GLUT4) expression in high glucose condition. So the aim of this in vivo study was to investigate whether L-carnitine can improve insulin resistance in type 2 diabetes (T2DM) through increasing the expression of GLUT 4 in skeletal muscles. Methods: Thirty healthy male Sprague Dawely rats were randomly divided into: Control negative, control positive (Diabetic) and Carnitine groups. For induction of T2DM the control positive and carnitine groups' rats were fed high fat diet (HFD) for about 2 month, while control negative rats were fed on normal diet. Diabetic and carnitine groups received 35 mg/kg streptozocin (STZ) intraperitoneal. After confirming the development of T2DM in diabetic and carnitine groups L-carnitine was administered orally to the carnitine group in a dose of 3 gm carnitine / kg once daily for 4 weeks. At the end of treatment period, rats were sacrificed and blood samples were collected directly from the heart and the hindlimb soleus muscles were removed, snap- frozen and stored at−80◦C until analysis. Results: L-carnitine administration resulted in significant decrease (p<0:05) in serum glucose, serum insulin, and triglycerides: High Density Lipoprotein Cholesterol (TG:HDL) ratio of the carnitine group as compared to diabetic group. Carnitine administration showed also significant increase AMP-activated protein kinase (AMPK) activity and in total skeletal muscle GLUT-4 protein concentration. Conclusion: The results of this study demonstrate that carnitine supplementation ameliorates insulin resistance in type 2 diabetic rats and up-regulating GLUT4 protein expression in the skeletal muscles through increasing the activity of AMPK. So, carnitine administration may be a possible candidate for the treatment of T2DM.