This work evaluated the cardioprotective effects of sonlicromanol, a new mitochondrial-directed drug, on cardiac ischemia/reperfusion (I/R) injury and explored the involvement of inflammatory and oxidative responses via activation of AMPK-eNOS-mitochondrial pathway. Male Sprague-Dawley rats underwent regional I/R injury through in vivo left anterior descending (LAD) coronary artery ligation for 40 minutes followed by 24 hours of reperfusion. Pretreatment of rats with sonlicromanol considerably reduced cardiac I/R injury in a dose-dependent manner, as indicated by lower infarct size and serum creatine-kinase levels, and improved cardiac function after reperfusion. Sonlicromanol (50 mg/kg) significantly reduced TNF-α, interleukin-1β, NF-κB-p65, and 8-isoprostane levels while increased manganese-superoxide dismutase and nitric-oxide levels and expression of eNOS and AMPK protein. It significantly reduced mitochondrial membrane depolarization and reactive oxygen species (ROS) levels. However, AMPK inhibition significantly reduced sonlicromanol protective actions. Cardioprotection by sonlicromanol was achieved by moderating inflammatory and oxidative responses, and AMPK/eNOS/mitochondrial signaling is a crucial regulator of these actions.