Background: Doxorubicin is an anthracycline antibiotic commonly used to treat a variety of cancers as a most effective antitumor. However, its clinical use is associated with the toxic effects in numerous healthy tissues. Here we investigated the pretreatment effect of regular aerobic exercise on oxidative stress in rats acutely exposed to DOX-induced hepatotoxicity. Materials and Methods: Forty-eight Wistar male rats were randomly divided into 2 groups: control and training. The training protocol included treadmill running between 25 to 54 min/day and 15 to 20m/min, 5 days/week for 6 weeks. At the end of the exercise training protocol, rats from the control and trained groups were again randomly separated into 3 subgroups: DOX10mg/kg, DOX20mg/kg and saline. All treatments were carried 24 h after the last exercise bout and animals were sacrificed 24 h after DOX and saline injections. Results: Administration of DOX (10 and 20 $mg.kg^{-1}$) resulted in imbalance in biomarkers related to oxidants and antioxidants in liver tissue, as compared to control groups. Six weeks of pretreatment training led to a significant increase in nitric oxide (NO), superoxide dismutase (SOD) and glutathione peroxidase (GPX) as compared to the control+DOX 10 mg/kg group. Training before DOX 20 mg/kg administration also led to a significant increase in NO and SOD, and a significant decrease in malondialdehyde (MDA). In addition, there was a significant difference between DOX 10 mg/kg and DOX 20 mg/kg treatments in MDA levels, only. Conclusions: The results of the present study indicate that pretreatment with aerobic exercise induces positive adaptations and has a potential protective effect against doxorubicin (DOX)-induced hepatotoxicity with doses of 10 and 20 mg.kg.