Calorie restriction (CR) alleviates insulin resistance and has a beneficial effect on numerous metabolic disorders, yet the underlying mechanism has not been fully elucidated. In the present study, we found that CR of mice (60% of the diet consumption compared with ad libitum mice) reduces the expression levels of Grb2 in skeletal muscle, an insulin target tissue that accounts for 85% of insulin-stimulated blood glucose clearance. To determine whether Grb2 down-regulation contributes to increased insulin sensitivity in the regulation of glucose metabolism, we generated C2C12 cell lines in which the expression of Grb2 is suppressed by RNA interference. Suppressing Grb2 expression in C2C12 myoblasts enhances insulin-stimulated IRS-1 tyrosine phosphorylation and Akt phosphorylation, which is associated with decreased IRS-1 serine phosphorylation at residues 307, 612, and 636/639. In addition, reducing Grb2 expression levels increased insulin-stimulated glucose uptake in C2C12 myotubes. Reduced IRS-1 serine phosphorylation is also found in Grb2+/- heterozygous knockout mice, which is associated with enhanced insulin signaling and resistance to high fat diet-induced glucose and insulin intolerance. All together, our results suggested that reducing the expression levels of Grb2 provides a mechanism by which CR increases insulin sensitivity in vivo.