Myocardial ischemia/reperfusion (MI/R) injury is a life-threatening disease with high morbidity and mortality. Herein, the present study is conducted to explore the regulatory mechanism of GSK3β in MI/R injury regarding cardiomyocyte apoptosis and oxidative stress. The MI/R injury mouse model and hypoxic reoxygenation (H/R) cell model were established. The expression pattern of GSK3β, FTO, KLF5, and Myc was determined followed by their relation validation. Next, loss-of-function experiments were implemented to verify the effect of GSK3β/FTO/KLF5/Myc on cardiomyocyte apoptosis and oxidative stress in the MI/R injury mouse model and H/R cell model. High expression of GSK3β and low expression of FTO, KLF5, and Myc were observed in the MI/R injury mouse model and H/R cell model. GSK3β promoted phosphorylation of FTO and KLF5, thus increasing the ubiquitination degradation of FTO and KLF5. A decrease of FTO and KLF5 was able to downregulate Myc expression, resulting in enhanced cardiomyocyte apoptosis and oxidative stress. These data together supported the crucial role that GSK3β played in facilitating cardiomyocyte apoptosis and oxidative stress so as to accelerate MI/R injury, which highlights a promising therapeutic strategy against MI/R injury.