Lipid infusion and high fat feeding are established causes of systemic and adipose tissue insulin resistance. In this study, we treated 3T3-L1 adipocytes with a mixture of free fatty acids (FFAs) to investigate the molecular mechanisms underlying fat-induced insulin resistance. FFA treatment impaired insulin receptor-mediated signal transduction and decreased insulin-stimulated GLUT4 translocation and glucose transport. FFAs activated the stress/inflammatory kinases c-Jun N-terminal kinase (JNK) and IKKbeta, and the suppressor of cytokine signaling protein 3, increased secretion of the inflammatory cytokine tumor necrosis factor (TNF)-alpha, and decreased secretion of adiponectin into the medium. RNA interference-mediated down-regulation of JNK blocked JNK activation and prevented most of the FFA-induced defects in insulin action. Blockade of TNF-alpha signaling with neutralizing antibodies to TNF-alpha or its receptors or with a dominant negative TNF-alpha peptide had a partial effect to inhibit FFA-induced cellular insulin resistance. We found that JNK activation by FFAs was not inhibited by blocking TNF-alpha signaling, whereas the FFA-induced increase in TNF-alpha secretion was inhibited by RNA interference-mediated JNK knockdown. Together, these results indicate that 1) JNK can be activated by FFAs through TNF-alpha-independent mechanisms, 2) activated JNK is a major contributor to FFA-induced cellular insulin resistance, and 3) TNF-alpha is an autocrine/paracrine downstream effector of activated JNK that can also mediate insulin resistance.