Abstract Accumulating evidence suggests that cytochrome P450 (CYP) epoxygenases metabolize arachidonic acid into epoxyeicosatrienoic acids (EETs) which play important roles in various pathophysiological processes. Interestingly, CYP-derived eicosanoids are vasodilatory, at least in part through their ability to activate eNOS and subsequent NO release. This study investigated the roles of eNOS in CYP2J3 gene delivery reducing blood pressure and improving insulin resistance in fructose-treated rats. CYP2J3 overexpression in vivo increased EET generation, reduced blood pressure and reversed insulin resistance as determined by insulin resistance index (HOMA-IR). Furthermore, administration of eNOS inhibitor L-NMMA significantly and partially abolished the beneficial effects of CYP2J3 gene delivery on hypertension and insulin resistance induced by fructose intake, and possible mechanism is associated with increased ET-1, ETA-receptor mRNA expression and reduced sensitivity of insulin to peripheral tissues and organs characterized by reduced activity of IRS-1/PI3K/AKT and AMPK signalling pathways. These data provide direct evidence that CYP2J3-derived EETs may alleviate insulin resistance at least in part through upregulated eNOS expression.