Numerous experimental studies have supported the evidence that 2-methoxyestradiol (2 ME) is a biologically active metabolite that mediates multiple effects on the cardiovascular system, largely independent of the estrogen receptor. 2 ME is a major cytochrome P450 1B1 (CYP1B1) metabolite and has been reported to have vasoprotective and anti-inflammatory actions. However, whether 2 ME would prevent cardiac hypertrophy induced by abdominal aortic constriction (AAC) has not been investigated yet. Therefore, the overall objectives of the present study were to elucidate the potential antihypertrophic effect of 2 ME and explore the mechanism(s) involved. Our results showed that 2 ME significantly inhibited AAC-induced left ventricular hypertrophy using echocardiography. The antihypertrophic effect of 2 ME was associated with a significant inhibition of CYP1B1 and mid-chain hydroxyeicosatetraenoic acids. Based on proteomics data, the protective effect of 2 ME is linked to the induction of antioxidant and anti-inflammatory proteins in addition to the modulation of proteins involved in myocardial energy metabolism. In vitro, 2 ME has shown a direct antihypertrophic effect through mitogen-activated protein kinases- and nuclear factor-κB-dependent mechanisms. The present work shows a strong evidence that 2 ME protects against left ventricular hypertrophy. Our data suggest the potential of repurposing 2 ME as a selective CYP1B1 inhibitor for the treatment of heart failure.