Palmitate has been shown to induce insulin resistance in skeletal muscle cells. The aim of this study was to investigate the role of the leukocyte common antigen-related (LAR) gene in palmitate-induced insulin resistance in C2C12 cells. A stable C2C12 cell line was generated using LAR short hairpin RNA. The levels of LAR protein and phosphorylation of insulin receptor substrate-1 (IRS1) and Akt were detected by western blot analysis. 2-Deoxyglucose uptake was measured in LAR knockdown and control cells using d-[2-(3)H]glucose. LAR protein level was decreased by 65% in the stable cell line compared with the control cells. Palmitate (0.5 mM) significantly induced LAR mRNA (65%) and protein levels (40%) in myotubes compared with untreated cells. Palmitate significantly reduced insulin-stimulated glucose uptake in both the control and LAR knockdown cells by 33 and 51% respectively. However, LAR depletion improved insulin-stimulated glucose uptake in myotubes treated with palmitate. Furthermore, the inhibition of LAR prevented palmitate-induced decreases in phosphorylation of IRS1(Tyr632) and Akt(Ser473) in C2C12 cells. In conclusion, these results reveal that palmitate induces LAR expression in C2C12 cells. We also provided evidence that the inhibition of LAR attenuates palmitate-induced insulin resistance in myotubes.