Protectin DX (PDX), a double lipoxygenase derivative of docosahexaenoic acid, has been reported to attenuate inflammation and insulin resistance. In the current study, we explored the effects of PDX on hyperlipidemia-induced insulin resistance and inflammation through AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor α (PPARα). PDX attenuated the impairment of insulin receptor substrate 1/Akt-mediated insulin signaling in palmitate-treated differentiated C2C12 cells and soleus skeletal muscle of HFD-fed mice. Furthermore, PDX treatment significantly ameliorated HFD-induced weight gain and improved glucose tolerance in mice. Nuclear factor kB nuclear translocation, inhibitory kBα phosphorylation, and expression of proinflammatory cytokines were markedly attenuated by PDX in both in vitro and in vivo models. PDX treatment markedly augmented AMPK phosphorylation and PPARα expression in C2C12 cells and in skeletal muscle of mice. AMPK- and PPARα-specific siRNAs significantly abrogated the suppressive effects of PDX on palmitate-induced insulin resistance and inflammation. Furthermore, PDX markedly stimulated the expression of genes related to fatty acid oxidation. These effects of PDX were significantly suppressed by AMPK and PPARα siRNAs. In conclusion, our results demonstrate that PDX ameliorates insulin resistance and inflammation and stimulates fatty acid oxidation through AMPK- and PPARα-mediated pathways in skeletal muscle.