Several reports demonstrated the direct contribution of cytochrome P450 1B1 (CYP1B1) enzyme and its associated cardiotoxic mid-chain, hydroxyeicosatetraenoic acid (HETEs) metabolites in the development of cardiac hypertrophy. Resveratrol is commercially available polyphenol that exerts beneficial effects in wide array of cardiovascular diseases including cardiac hypertrophy, myocardial infarction and heart failure. Nevertheless, the underlying mechanisms responsible for these effects are not fully elucidated. Since resveratrol is a well-known CYP1B1 inhibitor, the purpose of this study is to test whether resveratrol attenuates angiotensin II (Ang II)-induced cellular hypertrophy through inhibition of CYP1B1/mid-chain HETEs mechanism. RL-14 and H9c2 cells were treated with vehicle or 10 μM Ang II in the absence and presence of 2, 10 or 50 μM resveratrol for 24 h. Thereafter, the level of mid-chain HETEs was determined using liquid chromatography-mass spectrometry (LC/MS). Hypertrophic markers and CYP1B1 gene expression and protein levels were measured using real-time PCR and Western blot analysis, respectively. Our results demonstrated that resveratrol, at concentrations of 10 and 50 μM, was able to attenuate Ang-II-induced cellular hypertrophy as evidenced by substantial inhibition of hypertrophic markers, β-myosin heavy chain (MHC)/α-MHC and atrial natriuretic peptide. Ang II significantly induced the protein expression of CYP1B1 and increased the metabolite formation rate of its associated mid-chain HETEs. Interestingly, the protective effect of resveratrol was associated with a significant decrease of CYP1B1 protein expression and mid-chain HETEs. Our results provided the first evidence that resveratrol protects against Ang II-induced cellular hypertrophy, at least in part, through CYP1B1/mid-chain HETEs-dependent mechanism.