The p66^Shc protein isoform regulates MAP kinase activity and the actin cytoskeleton turnover, which are both required for normal glucose transport responses. To investigate the role of p66^Shc in glucose transport regulation in skeletal muscle cells, L6 myoblasts with antisense-mediated reduction (L6/p66^Shcas) or adenovirus-mediated overexpression (L6/p66^Shcadv) of the p66^Shc protein were examined. L6/^Shcas myoblasts showed constitutive activation of Erk-1/2 and disruption of the actin network, associated with an 11-fold increase in basal glucose transport. GLUT1 and GLUT3 transporter proteins were 7-fold and 4-fold more abundant, respectively, and were localized throughout the cytoplasm. Conversely, in L6 myoblasts overexpressing p66^Shc, basal glucose uptake rates were reduced by 30%, in parallel with a ~50% reduction in total GLUT1 and GLUT3 transporter levels. Inhibition of the increased Erk-1/2 activity with PD98059 in L6/^Shcas cells had a minimal effect on increased GLUT1 and GLUT3 protein levels, but restored the actin cytoskeleton, and reduced the abnormally high basal glucose uptake by 70%. In conclusion, p66^Shc appears to regulate the glucose transport system in skeletal muscle myoblasts by controlling, via MAP kinase, the integrity of the actin cytoskeleton and by modulating cellular expression of GLUT1 and GLUT3 transporter proteins via Erk-independent pathways.