Orientin, isolated from bamboo leaves, is an important natural antioxidant. It has been identified that orientin could protect myocardium against ischemia/reperfusion (I/R) injury. But the precise mechanism of this protective effect is still elusive. The ubiquitin-proteasome system (UPS) plays a central role in regulating a variety of critical cellular processes. Recently, the cardiac UPS has become increasingly recognized as the key regulator of cardiac function under both physiological and pathological conditions. Previous studies indicated that alterations in the UPS function are involved in the pathogenesis and progression of a variety of cardiac diseases including myocardial ischemia/reperfusion. The proteasome is the proteolytically active core of UPS and could cause various diseases when it malfunctions. Accordingly, the proteasome has become an attractive target for pharmaceutical interventions. In addition, the relationship of orientin and UPS in mediating I/R-induced cardiomyocytes injury is still unclear. Here, we used H9c2 cardiomytocytes to explore the relationship of orientin-induced cardioprotection and proteasome inhibition during I/R process. Our findings suggested that orientin could reduce H9c2 cells apoptosis subjected to I/R injury partly through surpressing the proteasome inhition. Further investigation revealed that this effect was associated with orientin-attenuated reactive oxygen species (ROS) generation and depolarization of mitochondrial membrane potential (Δψm). Orientin also enhanced Bcl-2 protein level, decreased the expression levels of Bax and Smac/DIABLO. Our results also indicated that this beneficial effect of orientin on Bax expression was dramatically attenuated by MG132, the proteasomal inhibitor. In summary, we demonstrated that orientin exerts protective functions in H9c2 cardiomytocytes against I/R-induced apoptosis partially through suppressing proteasome inhibition, and mitochondrial apoptotic pathway is probably involved in this effect.