This study examined sex differences in the regulation of pro- and anti-apoptotic proteins in cardiac hypertrophy and heart failure due to volume overload induced by arteriovenous (AV) shunt in rats. General characteristics and hemodynamic assessment revealed the presence of cardiac hypertrophy at 4 weeks of AV shunt in male (n = 12) and female (n = 12) rats, whereas heart failure was seen at 16 weeks in male rats only. Although a decrease in apoptosis was seen in hearts of both sexes at 4 weeks, an increase in apoptosis in males and a reduction in the female heart were observed at 16 weeks of AV shunt. Unlike females, increases in the pro-apoptotic proteins, BAX, caspases 3 and 9 were seen in 16 weeks post-AV shunt in male rats. While an increase in phospho-Bad was detected, phospho-Bcl-2 protein was decreased in males. Females showed an increase in only phospho-Bcl-2 protein at 16 weeks post-AV shunt. Ovariectomy (n = 12) abolished the increase in phospho-Bcl-2 protein, but this was restored by treatment with 17-β estradiol. These data suggest that downregulation of phospho-Bcl-2 and an upregulation of BAX may play a major role in cardiomyocyte apoptosis in heart failure due to volume overload in male rats. Furthermore, upregulation of phospho-Bcl-2 in the heart due to estrogen may confer resistance against cardiomyocyte apoptosis in females.