Reperfusion therapy is the most important method for treating acute myocardial infarction. However, myocardial ischemia reperfusion injury (MIRI) can offset the benefit of reperfusion therapy and worsen the outcome. In both ischemia and reperfusion, autophagy remains problematic. Activating molecule in Beclin1-regulated autophagy (Ambra1) is an important protein in autophagy regulation, and its function in MIRI remains unclear. Thus, we used H9C2 cells to investigate the function of Ambra1 in MIRI and the underlying mechanisms involved. Hypoxia and reoxygenation of H9C2 cells were used to mimic MIRI in vitro. During hypoxia, autophagy flux was blocked, then recovered in reoxygenation. Ambra1 overexpression increased autophagy in the H9C2 cells, as the LC3B II/I ratio increased, and alleviated cellular necrosis and apoptosis during hypoxia and reoxygenation. This effect was counteracted by an autophagy inhibitor. Knocking down Ambra1 can block autophagy which P62 sediment/supernatant ratio increased while the ratio of LC3B II/I decreased, and worsen outcomes. Ambra1 enhances autophagy in H9C2 cells by improving the stability and activity of the ULK1 complex. Reactive oxygen species (ROS) are an important cause of MIRI. ROS were reduced when Ambra1 was overexpressed and increased when Ambra1 was knocked down, indicating that Ambra1 can protect against hypoxia and reoxygenation injury in H9C2 cells by promoting autophagy and reducing ROS.