: Endocannabinoids are bioactive amides, esters, and ethers of long-chain polyunsaturated fatty acids. Evidence suggests that activation of the endocannabinoid pathway offers cardioprotection against myocardial ischemia, arrhythmias, and endothelial dysfunction of coronary arteries. As cardiac hypertrophy is a convergence point of risk factors for heart failure, we determined a role for endocannabinoids in attenuating endothelin-1-induced hypertrophy and probed the signaling pathways involved. The cannabinoid receptor ligand anandamide and its metabolically stable analog, R-methanandamide, suppressed hypertrophic indicators including cardiomyocyte enlargement and fetal gene activation (ie, the brain natriuretic peptide gene) elicited by endothelin-1 in isolated neonatal rat ventricular myocytes. The ability of R-methanandamide to suppress myocyte enlargement and fetal gene activation was mediated by CB2 and CB1 receptors, respectively. Accordingly, a CB2-selective agonist, JWH-133, prevented only myocyte enlargement but not brain natriuretic peptide gene activation. A CB1/CB2 dual agonist with limited brain penetration, CB-13, inhibited both hypertrophic indicators. CB-13 activated AMP-activated protein kinase (AMPK) and, in an AMPK-dependent manner, endothelial nitric oxide synthase (eNOS). Disruption of AMPK signaling, using compound C or short hairpinRNA knockdown, and eNOS inhibition using L-NIO abolished the antihypertrophic actions of CB-13. In conclusion, CB-13 inhibits cardiomyocyte hypertrophy through AMPK-eNOS signaling and may represent a novel therapeutic approach to cardioprotection.