Aim To investigate the role of calcium- calmodulin dependent kinaseⅡand calcineurin in tumor necrosis factor α（TNF-α）-induced cardiomyocyte hypertrophy through Phosphatidylinositol 3-kinase pathways. Methods Intracellular free Ca²⁺ concentration was measured by laser confocal microscopy. The protein content was assayed with Lowry’s method. The cardiomyocytes volumes were measured by computer photograph analysis system. The expression of CaMKⅡδB and CaN was determined by western blot. Results ①LY294002, a selective PI3K inhibitor, significantly suppress the elevation of intracellular free Ca²⁺ concentration induced by TNF-α in cultured ventricular myocytes from the neonatal rat. The effect was similar to that of LY294002+2-APB（P>0.05）, but lower than LY294002+ryanodine（P<0.05）. ②LY294002 significantly reduced the enhancements in protein content and cell size induced by TNF-α. The effect was similar to that of 2-APB+LY294002, but higher than 2-APB and lower than ryanodine+LY294002. LY294002, a PI3K inhibitor, suppressed the expression of CaMKⅡδB and CaN induced by TNF-α in myocytes which was similar to that of LY294002+2-APB. Conclusion TNF-α induced cardiac hypertrophy through activing PI3-kinase pathway in cultured ventricular myocytes from the neonatal rat, which was mediated partly by IP3R to mediate expression of CaMKII and calcineurin through increasing the intercellular Ca²⁺.