Introduction: Insulin resistance plays a major role in type 2 diabetes and obesity. In this disorder, lipid accumulation is accompanied by increased TNF-α expression in the muscle. The aims of this study were to evaluate the effects of tumor necrosis factor-α (TNF-α) gene knockdown on key elements of insulin signaling pathway (IRS-1 and Akt) and insulin resistance in C2C12 muscle cells in the presence and absence of palmitate. Materials and Methods: To knockdown protein expression of TNF-α, the C2C12 cells were transfected with the shRNA containing antisense sequence of murine TNF-α gene. The analysis of TNF-α protein expression and phosphorylation and protein levels of IRS-1and Akt were subsequently detected by western blot. Results: In TNF-α knockdown cells, the protein expression level of TNF-α was reduced by 58%. Under treatment with palmitate, insulin stimulated phosphorylation of IRS-1 (Tyr632) and Akt (Ser473) in knockdown cells was increased 1.7 and 2.6 fold, respectively, compared to the controls. Conclusions: Our findings showed that decreasing the TNF-α protein level can enhance the activity of the important elements of insulin signaling pathway (IRS-1 and Akt), leading to the improvement of insulin resistance in myotubes, data suggesting that TNF-α may potentially be a therapeutic target for fatty acid induced insulin resistance observed in type 2 diabetes and metabolic syndrome.