Acute myocardial infarction (AMI) is one of the major causes of morbidity and mortality in the world. Ischemia/reperfusion (I/R) injury-induced cardiomyocytes death is the main obstacle that limits the heart function recovery of the AMI patients. Reactive oxygen species (ROS) generated by mitochondria is the main pathological stimulus of cardiomyocytes death during heart I/R injury process. Hence, to understand the underlying mechanism of cardioymocytes proliferation and apoptosis under oxidative stress is crucial for effective AMI therapy. In this study, we found that the expression of long non-coding RNA HOTAIR was significantly downregulated in H9c2 cells in response to oxidative stimuli. HOTAIR knockdown further attenuated H9c2 cells proliferation and accelerated H9c2 cells apoptosis in oxidative stress, while HOTAIR overexpression can protect H9c2 cells from oxidative stress-induced injury. Additionally, HOTAIR acted as a sponge for miR-125. MiR-125 inhibitors restored the H9c2 cells proliferation and migration potential after HOTAIR knockdown in oxidative stress. Meanwhile, MMP2 was identified as a target of miR-125. MMP2 knockdown blocked miR-125 inhibitors' protect effect on H9c2 cells in oxidative stress. Further study demonstrated that HOTAIR inhibition can aggravate oxidative stress-induced H9c2 cells injury through HOTAIR/miR-125/MMP2 axis. Our finding revealed a novel regulatory mechanism for cardiomyocytes proliferation and apoptosis under oxidative stress conditions, which provided a therapeutic approach for myocardium repair after AMI injury.