Sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA2a) plays an essential role in Ca(2+) homeostasis and cardiac functions. The promoter region of SERCA2a has a high content of CpG islands; thus, epigenetic modification by inhibiting methylation can enhance SERCA2a expression in cardiomyocytes. Hydralazine, a drug frequently used in heart failure, is a potential DNA methylation inhibitor. We evaluated whether hydralazine can modulate Ca(2+) handling through an increase in SERCA2a expression via regulating methylation. We used indo-1 fluorescence, real-time RT-PCR, immunoblotting, and methylation-specific PCR to investigate intracellular Ca(2+), the expressions of RNA and protein, and methylation of SERCA2a in HL-1 cardiomyocytes with and without (control) the administration of hydralazine (1, 10, and 30 μM) for 72 h. Hydralazine (10 and 30 μM) increased the intracellular Ca(2+) transients and SR Ca(2+) contents. Hydralazine (10 and 30 μM) decreased methylation in the SERCA2a promoter region and increased the RNA and protein expressions of SERCA2a. Additionally, hydralazine (10 and 30 μM) decreased the expression of DNA methyltransferase 1. Moreover, treatment with hydralazine in isoproterenol-induced heart failure rats decreased the promoter methylation of SERCA2a and increased SERCA2a RNA expression. In conclusion, hydralazine-induced promoter demethylation may improve cardiac function through increasing SERCA2a and modulating calcium homeostasis in cardiomyocytes.