Physiological stressors such as sepsis and tissue damage initiate an acute immune response and cause transient systemic insulin resistance. This study was conducted to determine whether tumor necrosis factor-α (TNF-α), a cytokine produced by immune cells during skeletal muscle damage, decreases insulin responsiveness at the cellular level. To examine the molecular mechanisms associated with TNF-α and insulin action, we measured insulin receptor substrate (IRS)-1- and IRS-2-mediated phosphatidylinositol 3-kinase (PI 3-kinase) activation, IRS-1-PI 3-kinase binding, IRS-1 tyrosine phosphorylation, and the phosphorylation of two mitogen-activated protein kinases (MAPK, known as p42^MAPK and p44^MAPK) in cultured C₂C₁₂myotubes. Furthermore, we determined the effects of TNF-α on insulin-stimulated 2-deoxyglucose (2-DG) uptake. We observed that TNF-α impaired insulin stimulation of IRS-1- and IRS-2-mediated PI 3-kinase activation by 54 and 55% (P< 0.05), respectively. In addition, TNF-α decreased insulin-stimulated IRS-1 tyrosine phosphorylation by 40% (P < 0.05). Furthermore, TNF-α repressed insulin-induced p42^MAPKand p44^MAPK tyrosine phosphorylation by 81% (P < 0.01). TNF-α impairment of insulin signaling activation was accompanied by a decrease (P < 0.05) in 2-DG uptake in the muscle cells (60 ± 4 vs. 44 ± 6 pmol ⋅ min⁻¹ ⋅ mg⁻¹). These data suggest that increases in TNF-α may cause insulin resistance in skeletal muscle by inhibiting IRS-1- and IRS-2-mediated PI 3-kinase activation as well as p42^MAPK and p44^MAPK tyrosine phosphorylation, leading to impaired insulin-stimulated glucose uptake.