The pregnancy hormone relaxin has recently been shown to be cardio-protective. Despite its well-established anti-fibrotic actions in the heart, the effects of relaxin on cardiomyocytes (CM) remain to be determined. We investigated effects of isoform 2 of the human relaxin (H2-relaxin) on CM hypertrophy and apoptosis. In cultured neonatal rat CM, phenylephrine (50 µM) and cardiac fibroblast-conditioned medium (FCM) were used respectively to induce CM hypertrophy. The degree of hypertrophy was indicated by increased cell size, protein synthesis and gene expression of atrial natriuretic peptide (ANP). Whilst H2-relaxin (16.7 nM) alone failed to suppress hypertrophy induced by phenylephrine, it repressed the FCM-induced increase in protein synthesis by 24% (P<0.05) and reversed the increase in cell size (P<0.001) and ANP expression (P<0.01). We further studied the effect of H2-relaxin on CM apoptosis induced by H₂O₂ (200 µM). Studies of DNA laddering and nuclear staining demonstrated that H2-relaxin treatment reduced H₂O₂-induced DNA fragmentation. Real time PCR and Western blot analysis revealed a significant increase in the Bcl2/Bax ratio in H2-relaxin-treated CM. Further analysis showed that activation of Akt (1.8-fold, P<0.001) and ERK (2.0-fold, P<0.01) were involved in the anti-apoptotic action of H2-relaxin in CM, and that Gi/o coupling of relaxin receptors was associated with the H2-relaxin-induced Akt activation in CM. In conclusion, these results extend our current knowledge of the cardiac actions of relaxin by demonstrating that H2-relaxin indirectly inhibits CM hypertrophy and directly protects CM from apoptosis.