20-hydroxyeicosatetraenoic acid (20-HETE) has been shown to positively correlate with body mass index, hyperglycemia and plasma insulin levels. This study seeks to identify a causal relationship between 20-HETE and obesity-driven insulin resistance. Cyp4a14(-/-) male mice, a model of 20-HETE overproduction, were fed a regular or high-fat diet (HFD) for 15 weeks. 20-SOLA, a 20-HETE antagonist, was administered from week 0 or week 7 of HFD. HFD-fed mice gained significant weight (16.7+/-3.2 vs. 3.8+/-0.35 g, p<0.05) and developed hyperglycemia (157+/-3 vs 121+/-7 mg/dl, p<0.05) and hyperinsulinemia (2.3+/-0.4 vs 0.5+/-0.1 ng/ml, p<0.05) compared to regular diet-fed mice. 20-SOLA attenuated HFD-induced weight gain (9.4+/-1 vs 16.7+/-3 g, p<0.05), and normalized the hyperglycemia (157+/-7 vs 102+/-5 mg/dl, p<0.05) and hyperinsulinemia (1.1+/-0.1 vs 2.3+/-0.4 ng/ml, p<0.05). The impaired glucose homeostasis and insulin resistance in HFD-fed mice evidenced by reduced insulin and glucose tolerance were also ameliorated by 20-SOLA. Circulatory and adipose tissue 20-HETE levels significantly increased in HFD-fed mice correlating with impaired insulin signaling, including reduction in insulin receptor tyrosine (Y972) phosphorylation and increased serine (S307) phosphorylation of the insulin receptor substrate-1 (IRS-1). 20-SOLA treatments prevented changes in insulin signaling. These findings indicate that 20-HETE contributes to HFD-induced obesity, insulin resistance and impaired insulin signaling.