The mechanisms responsible for increased expression of TNF-α in skeletal muscle cells in diabetic states are not well understood. We examined the effects of the saturated acid palmitate on TNF-α expression. Exposure of C2C12 skeletal muscle cells to 0.75 mm palmitate enhanced mRNA (25-fold induction, P < 0.001) and protein (2.5-fold induction) expression of the proinflammatory cytokine TNF-α. This induction was inversely correlated with a fall in GLUT4 mRNA levels (57% reduction, P < 0.001) and glucose uptake (34% reduction, P < 0.001). PD98059 and U0126, inhibitors of the ERK-MAPK cascade, partially prevented the palmitate-induced TNF-α expression. Palmitate increased nuclear factor (NF)-κB activation and incubation of the cells with the NF-κB inhibitors pyrrolidine dithiocarbamate and parthenolide partially prevented TNF-α expression. Incubation of palmitate-treated cells with calphostin C, a strong and specific inhibitor of protein kinase C (PKC), abolished palmitate-induced TNF-α expression, and restored GLUT4 mRNA levels. Palmitate treatment enhanced the expression of phospho-PKCθ, suggesting that this PKC isoform was involved in the changes reported, and coincubation of palmitate-treated cells with the PKC inhibitor chelerythrine prevented the palmitate-induced reduction in the expression of IκBα and insulin-stimulated Akt activation. These findings suggest that enhanced TNF-α expression and GLUT4 down-regulation caused by palmitate are mediated through the PKC activation, confirming that this enzyme may be a target for either the prevention or the treatment of fatty acid-induced insulin resistance.