Rhodiola rosea improves exercise endurance and fatigue. We hypothesized that ingredients in Rhodiola rosea may increase antioxidant capability against swimming induced oxidative stress. In this study, we have identified the Rhodiola rosea ingredients, p-tyrosol, salidroside, rosin, rosavin and rosarin by high performance liquid chromatography-mass spectrometer and evaluated their O2(-)*, H2O2, and HOCl scavenging activities by a chemiluminescence analyzer. We next explored the effect and mechanism of Rhodiola rosea on 90-min swimming-induced oxidative stress in male Wistar rats fed with three doses of Rhodiola rosea extracts in drinking water (5, 25, 125 mg/day/rat) for 4 weeks. Our results showed that the 4 major ingredients (salidroside, rosin, rosavin and rosarin) from Rhodiola rosea extracts scavenged O2(-)*, H2O2, and HOCl activity in a dose-dependent manner. The ninety-min swimming exercise increased the O2(-)* production in the order: liver > skeletal muscle > blood, indicating that liver is the most sensitive target organ. The level of plasma malonedialdehyde, a lipid peroxidation product, was also increased after exercise. Treatment of 4 weeks of Rhodiola rosea extracts significantly inhibited swimming exercise-enhanced O2(-)* production in the blood, liver and skeletal muscle and plasma malonedialdehyde concentration. The expression in Mn-superoxide dismutase Cu/Zn-superoxide dismutase, and catalase in livers were all enhanced after 4 weeks of Rhodiola rosea supplementation especially at the dose of 125 mg/day/rat. Treatment of Rhodiola rosea extracts for 4 weeks significantly increased swimming performance. In conclusion, treatment of Rhodiola rosea extracts for 4 weeks could reduce swimming-enhanced oxidative stress possibly via the reactive oxygen species scavenging capability and the enhancement of the antioxidant defense mechanisms.