We have shown that Alox15, the gene encoding for 12/15‐lipoxygenase (12/15‐LO), is markedly up‐regulated in livers from apolipoprotein E‐deficient (ApoE^−/−) mice, which spontaneously develop nonalcoholic fatty liver disease secondary to hyperlipidemia. In the current study, we used ApoE^−/− mice with a targeted disruption of the Alox15 gene to assess the role of 12/15‐LO in the development and progression of hepatic steatosis and inflammation. Compared with ApoE^−/− mice, which exhibited extensive hepatic lipid accumulation and exacerbated inflammatory injury, ApoE/12/15‐LO double‐knockout (ApoE^−/−/12/15‐LO^−/−) mice showed reduced serum alanine aminotransferase levels; decreased hepatic steatosis, inflammation, and macrophage infiltration; and decreased fatty acid synthase, tumor necrosis factor α (TNFα), monocyte chemoattractant protein‐1 (MCP‐1), interleukin (IL)‐18, and IL‐6 expression. Remarkably, disruption of Alox15 attenuated glucose intolerance and high‐fat diet‐induced insulin resistance, up‐regulated insulin receptor substrate‐2, and exerted opposite effects on hepatic c‐Jun amino‐terminal kinase and adenosine monophosphate–activated protein kinase phosphorylation, known negative and positive regulators of insulin signaling, respectively. In adipose tissue, the absence of Alox15 induced significant reductions in the expression of the proinflammatory and insulin‐resistant adipokines MCP‐1, TNFα, and resistin while increasing the expression of glucose transporter‐4. Interestingly, compared with ApoE^−/− mice, which exhibited increased hepatic caspase‐3 staining, ApoE^−/−/12/15‐LO^−/− mice showed attenuated hepatocellular injury. Consistent with this finding, hepatocytes isolated from ApoE^−/− mice were more vulnerable to TNFα‐induced programmed cell death, an effect that was not observed in hepatocytes carrying a targeted disruption of the Alox15 gene. Conclusion: Collectively, our data suggest a potentially relevant mechanism linking 12/15‐LO to the promotion of hepatic steatosis, insulin resistance, and inflammation in experimental liver disease of metabolic origin. (HEPATOLOGY 2010)