BACKGROUND: Branched-chain amino acids, especially leucine, are known to interact with insulin signaling pathway and glucose metabolism. However, the mechanism by which this is exerted, remain to be clearly defined. In order to examine the effect of leucine on muscle insulin signaling, a set of experiments was carried out to quantitate phosphorylation events along the insulin signaling pathway in human skeletal muscle cell cultures. Cells were exposed to insulin, leucine or both, and phosphorylation events of key insulin signaling molecules were tracked over time so as to monitor time-related responses that characterize the signaling events and could be missed by a single sampling strategy limited to pre/post stimulus events. RESULTS: Leucine is shown to increase the magnitude of insulin-dependent phosphorylation of protein kinase B (AKT) at Ser473 and glycogen synthase kinase (GSK3ß) at Ser21-9. Glycogen synthesis follows the same pattern of GSK3ß, with a significant increase at 100 µM leucine plus insulin stimulus. Moreover, data do not show any statistically significant increase of pGSK3ß and glycogen synthesis at higher leucine concentrations. Leucine is also shown to increase the magnitude of insulin-mediated extracellularly regulated kinase (ERK) phosphorylation; however, differently from AKT and GSK3ß, ERK shows a transient behavior, with an early peak response, followed by a return to the baseline condition. CONCLUSIONS: These experiments demonstrate a complementary effect of leucine on insulin signaling in a human skeletal muscle cell culture, promoting insulin-activated GSK3ß phosphorylation and glycogen synthesis.