Background: Females have a lower incidence of heart failure and improved survival after myocardial ischemia/reperfusion (I/R) versus males. Although estrogen-suppressed cardiomyocyte apoptosis may be mediated through the PI3K/Akt, it is unclear whether this action is mediated via estrogen receptor beta (ERb). Therefore, we hypothesized that ERb mediates estrogen-induced cardioprotection through PI3K/Akt and anti-apoptotic signaling in females, but not males. Methods and Results: Isolated male and female hearts from ERb knockout (ERbKO) and wild type (WT) mice (n=5/group) were subjected to 20-minute ischemia followed by 60-minute reperfusion (Langendorff). Ablation of ERb significantly decreased post-ischemic recovery of LVDP in female hearts, but not males. Reduced activation of PI3K and Akt was noted in female ERbKO hearts, which was associated with increased expression of caspase-3 and -8, as well as decreased Bcl-2 levels compared to WT. However, ERbKO did not change myocardial STAT3, SOCS3, VEGF, TNFR1 and TNFR2 in either gender following I/R. Furthermore, deficiency of ERb increased myocardial JNK activation in females, but increased ERK1/2 activity in males during acute I/R. Conclusions: ERb mediates myocardial protection via up-regulation of PI3K/Akt activation, decreased caspase-3, -8, and increased Bcl-2 in female hearts following I/R. These findings provide evidence of ERb-mediated PI3K/Akt and anti-apoptotic signaling in the myocardium, and may lend insight into the mechanistic pathways behind the observed variation in clinical outcomes between males and females after MI.