Insulin resistance is a cardinal feature of Type 2 Diabetes (T2D), which accompanied by lipid accumulation and TNF-alpha overexpression in skeletal muscle. The role of TNF-alpha in palmitate-induced insulin resistance remained to be elucidated. Here, we assessed effects of TNF-alpha knockdown on the components of insulin signaling pathway (IRS-1 and Akt) in palmitate-induced insulin resistant C2C12 skeletal muscle cells. To reduce TNF-a expression, C2C12 cells were transduced with TNF-alpha-shRNA lentiviral particles. Afterwards, the protein expression of TNF-alpha, IRS-1, and Akt, as well as phosphorylation levels of IRS-1 and Akt were evaluated by western blot. We also measured insulin-stimulated glucose uptake in the presence and absence of palmitate. TNF-alpha protein expression in C2C12 cells significantly increased by treatment with 0.75 mM palmitate (P < 0.05). In TNF-alpha knockdown cells, the protein expression level of TNF-alpha was significantly decreased by almost 70% (P < 0.01) compared with the control cells. Our results also revealed that, in control cells, palmitate treatment significantly reduced the insulin-induced phosphorylations of IRS-1 (Tyr632) and Akt (Ser473) by 60% and 66% (P < 0.01), respectively. Interestingly, these phosphorylations, even in the presence of palmitate, were not significantly reduced in TNF-alpha knockdown cells with respect to the untreated control cells (P > 0.05). Furthermore, palmitate significantly reduced insulin-dependent glucose uptake in control cells, however, it was not able to reduce insulin-stimulated glucose uptake in TNF-alpha knockdown cells in comparison with the untreated control cells (P < 0.01). These findings indicated that TNF-alpha down-regulation maintains insulin sensitivity, even in the presence of palmitate, therefore, TNF-alpha inhibition could be a good strategy for the treatment of palmitate-induced insulin resistance. (C) 2015 Elsevier Inc. All rights reserved.