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, and mitochondrial pathway might be involved in this effect. But the precise mechanism underlying this protective effect is still elusive. Mitochondrial channels are proved to be the important effectors of cell life and death. Especially, mitochondrial calcium uniporter (MCU) has shown particular contribution to cardiomyocytes under specific pathological or physiological conditions. The role of MCU in regulating I/R-induced heart injury is a novel research area. In addition, the relationship of orientin and MCU in mediating reperfusion-induced cardiomyocytes injury is still elusive. In the present study, we used H9c2 cardiomytocytes to investigate the effect of orientin on MCU during reperfusion. Our results indicated that orientin could prevent the MCU opening in H9c2 cells subjected to I/R injury. Further investigation revealed that this effect was correlated with orientin-attenuated reactive oxygen species (ROS) production, depolarization of mitochondrial membrane potential (Δψm), mitochondrial cytochrome c release and mitochondrial Ca2+ accumulation. Our results suggested that these beneficial effects of orientin were partially blocked by spermine, an activator of MCU. In summary, the findings indicate that orientin protects H9c2 cardiomytocytes against ischemia/reperfusion injury via inhibiting mitochondrial calcium uniporter opening，and PI3K/Akt signaling pathway may be involved in these effects of orientin.