Apoptosis of cardiomyocytes, under ischemic conditions, has been identified as an essential process in the progression of heart failure. Under hypoxic conditions, mitochondria can become a threat to the cell because of their capacity to generate reactive oxygen species (ROS). As ROS appear to have a critical role in heart failure, there has been considerable interest in identifying the candidate proteins involved and in developing strategies to reduce oxidative stress. Lon protease (Lon) is a multifunctional protein that mediates protein quality control and stress response in mitochondria. However, comprehensive and detailed studies, on the role of Lon in hypoxia-induced cardiomyocyte apoptosis, have yet to be carried out. In the present study, we demonstrated that hypoxia induced ROS-dependent cardiomyocyte apoptosis. Lon was upregulated in hypoxia-induced cardiomyocytes. Lon downregulation attenuated hypoxia-induced cardiomyocyte apoptosis through a reduction of ROS level. Moreover, overexpression of Lon stimulated ROS production and induced apoptosis under normoxic conditions in cardiomyocytes. Our results identify Lon as a novel regulator of cardiomyocyte fate and offers exciting new insights into the therapeutic potential of hypoxia-induced cardiomyocyte apoptosis.