This study investigated the effect of carbohydrate (CHO) ingestion on skeletal muscle creatine (Cr) accumulation during Cr supplementation in humans. Muscle biopsy, urine, and plasma samples were obtained from 24 males before and after ingesting 5 g Cr in solution (group A) or 5 g Cr followed, 30 min later, by 93 g simple CHO in solution (group B) four times each day for 5 days. Supplementation resulted in an increase in muscle phosphocreatine (PCr), Cr, and total creatine (TCr; sum of PCr and Cr) concentration in groups A and B, but the increase in TCr in group B was 60% greater than in group A (P < 0.01). There was also a corresponding decrease in urinary Cr excretion in group B (P < 0.001). Creatine supplementation had no effect on serum insulin concentration, but Cr and CHO ingestion dramatically elevated insulin concentration (P < 0.001). These findings demonstrate that CHO ingestion substantially augments muscle Cr accumulation during Cr feeding in humans, which appears to be insulin mediated.