Late remote ischemia preconditioning (RIPC) and electro-acupuncture (EA) have both been suggested to reduce injury caused by myocardial ischemia/reperfusion (I/R). Our previous study has found that cardioprotection in RIPC is STAT5-dependent. Here, we aim to observe the effects of electro-acupuncture pretreatment (EAP) on I/R in the presence or absence of STAT5 in mice and investigate whether the protection of EAP is in a STAT5-dependent manner. In this study, EAP decreased myocardial infarction size (IS) /total area (TA) and rate of cardiomyocyte apoptosis. STAT5 was activated by EAP in the Stat5fl/fl mice but not in the Stat5-cKO mice, whereas, STAT3 was activated by EAP only in the Stat5-cKO but not in the Stat5fl/fl mice. Differentially expressed genes (DEGs) regulated by EAP in the Stat5fl/fl and the Stat5-cKO mice were quite distinct, indicating that EAP may activate IL-6/STAT3 signal in the absence of Stat5, and that EAP-induced cardioprotection against myocardial I/R injury was correlated with the activation of anti-apoptotic signaling and cardiomyocyte-survival signaling. Our results, for the first time, demonstrated that the protective effect of EAP was attributed to, but not dependent on, STAT5.