The SLC7A11/GPX4 axis plays an important role in ferroptosis during cardiac ischemia/reperfusion injury (IRI). The present study was designed to evaluate the impact of dexmedetomidine (DEX) post-conditioning on cardiac IRI and to explore whether the effect was achieved by SLC7A11/GPX4 signaling pathway regulation. Rat myocardial IRI was established by occluding the left anterior descending artery for 30 min followed by 2-h reperfusion. The infarct area was detected by diphenyltetrazolium chloride (TTC) staining; the cardiac function was evaluated by echocardiography. The levels of lipid peroxide biomarkers were measured to estimate the injury caused by lipid peroxide. HE staining and Sirius staining were utilized to assess myocardial damage and fibrosis. The mitochondrial morphology was observed by electron micrography. Western blot and quantitative real-time polymerase chain reaction were employed to measure the relative molecular characteristics. Our results showed that DEX administration at the beginning of reperfusion attenuated IRI-induced myocardial injury, alleviated mitochondrial dysfunction, decreased the level of reactive oxygen species (ROS), alleviated mitochondrial dysfunction, inhibited the activation of SLC7A11/GPX4, and modulated the expression of ferroptosis-related proteins, including SLC7A11, glutathione peroxidase 4 (GPX4), ferritin heavy chain (FTH), and cyclooxygenase-2 (COX-2). Conversely, the ferroptosis activator erastin partly suppressed the DEX-mediated cardio protection. Altogether, these results reveal that DEX inhibits ferroptosis by enhancing the expression of SLC7A11 and GPX4, thereby preventing cardiac I/R injury.