Hypoxia-induced apoptosis and mitochondrial dysfunction in cardiomyocytes are involved in the mechanisms of heart failure. Our previous studies have confirmed that NRF-1 alleviates hypoxia-induced injury by promoting mitochondrial function and inhibiting apoptosis in cardiomyocytes. However, the mechanism by which NRF-1 attenuates hypoxia-induced injury in cardiomyocytes is still unclear. Mitophagy, a selective autophagy, has recently shown a remarkable correlation with hypoxia-induced cardiomyocyte injury. In this study, we evaluated whether NRF-1 protects cardiomyocytes from hypoxia-induced injury by regulating mitophagy. The findings indicate that hypoxia prevents H9c2 cells from growing, encourages mitochondrial dysfunction, and triggers mitophagy. In addition, promoting mitophagy by rapamycin reduces hypoxia-induced injury in H9c2 cells. Overexpression of NRF-1 in hypoxia-induced H9c2 cells promotes mitophagy and alleviates cell injury, and this effect can be inhibited by 3-MA. Further study found that NRF-1 promotes the expression of FUNDC1 by binding to its promoter region. Knockdown of FUNDC1 in NRF-1 over-expression H9c2 cells inhibited mitophagy and aggravated hypoxia-induced injury. In conclusion, our study suggests that NRF-1 protects against hypoxia-induced injury by regulating FUNDC1-mediated mitophagy in cardiomyocytes.