Background and aims: Chlorpyrifos (CPF) is an insecticide that is widely used in the world. The purpose of this research was to investigate the effect of 4-week aerobic exercise and eugenol supplementation on the phosphatidylinositol-3-kinases/protein kinase/mammalian target of rapamycin (Pi3K/AKT/mTOR) pathway on the skeletal muscle of male rats poisoned with CPF. Methods: Overall, 12-week-old female rats were used in this experimental research. The rats were randomly divided into 8 groups (8 rats in each group), including healthy control, toxic control, poison solvent, corn oil solvent, poisoned+eugenol, poisoned+aerobic exercise, and poisoned+aerobic exercise+eugenol. Moderate training was in the range of 50-60% VO2 max, including 5 training sessions per week (treadmill). Poisoning was performed with CPF poison with a dose of 3 mg/kg, and the dose of eugenol was determined to be 250 mg/kg. Results: There was no significant difference between the groups in terms of mTOR and AKT expression (P=0.369, P=0.59). However, the expression of PI3k in the poisoned control group was lower than that in the healthy control group (P=0.049). In addition, the expression of PI3k was higher in the poisoned+eugenol+exercise group compared to the poisoned control group (P=0.009). The corn solvent group also had a higher PI3k expression in comparison to the poisoned control group (P=0.025). Finally, there was no significant difference among the other groups. Conclusion: In general, 4 weeks of CPF poisoning caused a significant decrease in PI3K, but it did not have a significant effect on AKT and mTOR. Based on the finding, 4 aerobic exercises and eugenol consumption could significantly increase in PI3K, while it had no significant effect on AKT and mTOR.