Cell cycle arrest is associated with differentiation, senescence and apoptosis. We investigated alterations in the cell cycle during the development of hypertrophy induced by hydrogen peroxide (H₂O₂) in the H9c2 clonal myoblastic cell line. H₂O₂ induced hypertrophy in H9c2 cells that was indicated by an increase in atrial natriuretic peptide (ANP) gene expression, a marker of cardiomyocyte hypertrophy, and a larger cell size. On induction of hypertrophy by H₂O₂ in H9c2 cells, cell proliferation was arrested, indicated by the number of cells remaining constant during a 72-h incubation period. The cell cycle was arrested at the G1 and G2/M phases with an increase in p21 expression, a negative cell cycle regulator. Cell cycle arrest and increase in p21 expression were significantly inhibited by 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra (acetoxymethyl) ester (BAPTA-AM), an intracellular calcium chelator. Although ANP gene expression was induced significantly, H₂O₂ failed to induce hypertrophy in the presence of BAPTA-AM, and the cell cycle progressed. We concluded that H₂O₂ induced cell cycle arrest in H9c2 cells, which was related to cellular hypertrophy.