Spexin (SPX) is a novel peptide with pleiotropic functions in adipose tissue including energy balance adjustment, fatty acid uptake, and glucose homeostasis. SPX level is closely associated with cardiovascular risk factors such as age, obesity, hypertension, and diabetes; however, its physiological significance in the cardiovascular system remains mostly undefined. We therefore here investigated the roles of SPX in regulating hypoxia-induced alterations in energy metabolism and mitochondrial function. We firstly confirmed that SPX is expressed in human and mouse cardiac tissue and documented that exposure to hypoxia in vitro reduces SPX level in rat H9C2 cardiomyocytes and primary neonatal rat ventricular myocytes (NRVMs). We then treated primary NRVMs with SPX before exposure to hypoxia, which (1) promoted fatty acid metabolism by enhancing expression of FAT/CD36, CPT1, ACADM, and PPAR-a and PGC1-a; (2) did not improve impaired glucose uptake; and (3) significantly prevented the downregulation of TFAM and mitochondrial electron transport chain complex and restrained UCP2 level and reactive oxygen species (ROS) production, thus enhancing ATP level in cardiomyocytes. In summary, SPX protects energy and mitochondrial homeostasis of cardiomyocytes during hypoxia, thereby highlighting the potential importance of SPX in the treatment of cardiovascular diseases.