GSK3β is a promising target for treating various disease conditions, including myocardial ischemia–reperfusion injury (IR). This study investigated the potential of GSK3β as a novel drug for managing IR in rats exposed to PM_2.5 for 1 day and up to 21 days. Female Wistar rats were exposed to PM_2.5 at a concentration of 250 µg/m³ for 3 h daily for either a single day or 21 days. After exposure, the isolated rat hearts underwent 30 min of ischemia followed by 60 min of reperfusion. GSK3β inhibition effectively reduced IR injury in rat hearts from animals exposed to PM_2.5 for 1 day but not in those exposed for 21 days. PM_2.5 exposure disrupted the redox balance in mitochondria and reduced the gene expression of antioxidants (glutaredoxin and peroxiredoxin) and NRF2, which protects against oxidative stress. PM_2.5 also impaired mitochondrial bioenergetics, membrane potential, and quality control, leading to mitochondrial stress. Importantly, PM_2.5 increased the translocation of GSK3β into mitochondria and compromised the overall mitochondrial function, particularly in the 21-day-exposed rat myocardium. The results indicate that extended exposure to PM_2.5 leads to oxidative stress that disrupts mitochondrial function and diminishes the effectiveness of GSK3β inhibitors in offering cardio-protection through mitochondria.