The purpose of this study was to investigate the role of insulin on skeletal muscle GLUT4 protein expression and glycogen storage after post-exercise carbohydrate supplementation. Male Sprague Dawley rats were randomly assigned to one of six treatment groups: sedentary control (CON), sedentary control with streptozocin (STZ/C), immediately post-exercise (EX0), immediately post-exercise with STZ (STZ/EX0), 5-h post-exercise (EX5), and 5-h post-exercise with STZ (STZ/EX5). Rats were exercised by swimming (2 bouts of 3-h) and carbohydrate supplemented immediately after each exercise session by glucose intubation (1 ml of a 50% w/v). Streptozocin was administered 72-h before exercise, which resulted in hyperglycemia and elimination of the insulin response to the carbohydrate supplement. GLUT4 protein of EXO rats was 30% above CON in fast-twitch (FT) red and 21% above CON in FT white muscle. Five h post-exercise (EX5), GLUT4 protein was 52% above CON in FT red and 47% above CON in FT white muscle. Muscle glycogen in FT red and white muscle was also increased above CON in EX5 rats. Neither GLUT4 protein nor muscle glycogen was increased above CON in STZ/EXO or STZ/EX5 rats. GLUT4 mRNA in FT red muscle of EXO rats was 61% above CON, but only 33% above CON in EX5 rats. GLUT4 mRNA in FT red muscle of STZ/C and STZ/EXO rats was similar, but significantly elevated in EX5/STZ rats. These results suggest that insulin is essential for the increase in GLUT4 protein expression following post-exercise carbohydrate supplementation.