AS-1, the TIR/BB loop mimetic, plays a protective role in cardiac ischemia/reperfusion (I/R) but the molecular mechanism remains unclear. The muscle specific caveolin3 (Cav-3) and the caveolae have been found to be critical for cardioprotection. This study aimed to evaluate our hypothesis that caveolae and Cav-3 are essential for AS-1-induced cardioprotection against myocardial I/R injury. To address these issues, we analyzed the involvement of Cav-3 in AS-1 mediated cardioprotection both in vivo and in vitro. We demonstrate that AS-1 administration significantly decreased infarct size, improved cardiac function after myocardial I/R and modulated membrane caveolae and Cav-3 expression in the myocardium. For in vitro studies, AS-1 treatment prevented Cav-3 re-distribution induced by H/R injury. In contrast, disruption of caveolae by MCD treatment or Cav-3 knockdown abolished the protection against H/R-induced myocytes injury by AS-1. Our findings reveal that AS-1 attenuates myocardial I/R injury through caveolae and Cav-3 dependent mechanism.