Signal transducer and activator of transcription (STAT) 3 is involved in mediating a broad range of biological processes, including cell survival, proliferation and immune response. Recent evidence has indicated that STAT3 in cardiomyocytes can be activated by ischemic/oxidative stress, and exerts cardioprotection in ischemic heart. There is no information, however, regarding the effect of endothelial cell derived STAT3 on the myocardial response to ischemia and reperfusion (I/R) injury. We hypothesized that ablation of the STAT3 gene in endothelial cells would worsen post-ischemic myocardial function by affecting capillary network integrity, suppressing anti-apoptotic signaling. Isolated hearts from wild type and endothelial cell STAT3 knockout (STAT3KO) mice were subjected to 20 minutes of global ischemia followed by 60 minutes of reperfusion. Endothelial cell STAT3 deficiency decreased recovery of myocardial function in response to I/R, which was associated with higher levels of LDH release, decreased activation of myocardial STAT3, and elevated p38 MAPK activation in STAT3 endothelial KO hearts. In addition, although no significant apoptosis was observed in wild type and KO hearts, our results showed more expression of myocardial caspase-8 and more apoptosis in the myocardium around the capillary in STAT3KO mice subjected to I/R. Further, endothelial cell STAT3 ablation resulted in increased myocardial expression of IL-6 and suppressor of cytokine signal (SOCS) 3. This study demonstrates that endothelial cell derived STAT3 plays an important role in post-ischemic myocardial function.