Skeletal muscle is a major site of insulin action. Intramuscular lipid accumulation results in inflammation, which has a strong correlation with skeletal muscle insulin resistance (IR). The aim of this study was to explore the effects of linoleic acid, alpha-linolenic acid, and gamma-linolenic acid (GLA), 18-carbon polyunsaturated fatty acids (PUFAs), on palmitic acid (PA)-induced inflammatory responses and IR in C2C12 myotubes. Our data demonstrated that these three test 18-carbon PUFAs can inhibit PA-induced interleukin-6 and tumor necrosis factor-α messenger RNA (mRNA) expression and IR as evidenced by increases in phosphorylated AKT and the 160-kD AKT substrate, mRNA and plasma membrane protein expression of glucose transporter 4, and glucose uptake. Moreover, the 18-carbon PUFAs blocked the effects of PA on activation of mitogen-activated protein kinases (MAPKs), protein kinase C-θ (PKC-θ), AMP-activated protein kinase (AMPK) and nuclear factor-κB (NF-κB). Of note, supplementation with GLA-rich borage oil decreased proinflammatory cytokine production and hindered the activation of MAPKs, PKC-θ and NF-κB in the skeletal muscles of diabetic mice. The 18-carbon PUFAs did not reverse PA-induced inflammation or IR in C2C12 myotubes transfected with a constitutively active mutant IκB kinase-β plasmid, which suggests the importance of the inhibition of NF-κB activation by the 18-carbon PUFAs. Moreover, blockade of AMPK activation by short hairpin RNA annulled the inhibitory effects of the 18-carbon PUFAs on PA-induced IR but not inflammation. Our findings suggest that the 18-carbon PUFAs may be useful in the management of PA-induced inflammation and IR in myotubes.