Neuropeptide Y (NPY) has been shown to participate in cardiac hypertrophy. However, the mechanisms by which NPY induces cardiomyocyte hypertrophy are poorly understood. This study tested the hypothesis that NPY induces cardiomyocyte hypertrophy through Ca2+/CaM-dependent calcineurin (CaN) pathway in cultured neonatal rat cardiomyocytes. After 24-h treatment, NPY (100 nM) significantly increased 3H-leucine incorporation and c-Jun mRNA expression, concomitant with augment of CaN activity and protein level in cardiomyocytes compared to those cells without NPY treatment. The enhancement of 3H-leucine incorporation and c-Jun mRNA expression in cardiomyocytes treated with NPY were markedly inhibited by cyclosporine A (CsA), a selective inhibitor of CaN. We also investigated the effect of NPYon intracellular Ca2+ level in cardiomyocytes. There were no obvious changes in intracellular Ca2+ level of cytoplasm and nucleus in cardiomyocytes treated with NPY (100 nM) for 10 min. However, NPY significantly increased intracellular Ca2+ level of cytoplasm and nucleus in cardiomyocytes after 24-h treatment. The result suggested that NPY could induce hypertrophy of cardiomyocytes via Ca2+/CaMdependent CaN signal pathway. The enhancement of [Ca2+]i caused by NPY may activate CaN signal pathways to mediate cardiac hypertrophy.