The objective of this research was to evaluate the effects of betulinic acid (BA) on ameliorating dexamethasone (Dex)-induced oxidative damage and the mechanism for the BA-mediated antioxidative effects. Forty male healthy Kunming mice were randomly divided into 5 groups, which were control (NC) group, Dex group, 0.25 mg/kg BA group, 0.50 mg/kg BA group and 1.00 mg/kg BA group. NC and Dex groups were administered orally with 1% starch solution, and the other groups were administered orally with different doses of BA for 14 days. Except NC group, mice in the other groups were intraperitoneal injected Dex (25 mg/kg BW) to set up oxidative damage model. The total antioxidant capacity (T-AOC), ability of inhibiting hydroxyl radical and peroxidase (POD) activity in liver, spleen and thymus were determined. The gene expressions of apoptosis signal-regulating kinase 1 (ASK1), c-Jun N-terminal kinase (JNK) and P38 in spleen and thymus through mitogen-activated protein kinase (MAPK) signal transduction pathway were determined by RT-PCR. The protein expressions of ASK1, JNK and P38 in spleen through MAPK signal transduction pathway were determined by Western Blot. The results showed as follows:1) compared with NC group, the T-AOC, ability of inhibiting hydroxyl radical and POD activity in liver, and POD activity in spleen, and T-AOC and ability of inhibiting hydroxyl radical in thymus of Dex group were significantly decreased (P<0.01). Compared with Dex group, the T-AOC, ability of inhibiting hydroxyl radical and POD activity in liver of 0.50 and 1.00 mg/kg BA groups were significantly increased (P<0.05 or P<0.01); the T-AOC in spleen of 0.50 mg/kg BA group, the ability of inhibiting hydroxyl radical in spleen of 0.50 and 1.00 mg/kg BA groups, and the POD activity in spleen of 0.25 and 1.00 mg/kg BA groups were significantly increased (P<0.05 or P<0.01); the T-AOC, ability of inhibiting hydroxyl radical and POD activity in thymus of 0.50 and 1.00 mg/kg BA groups were significantly increased (P<0.05 or P<0.01). 2) Compared with NC group, the mRNA expressions of ASK1, JNK and P38 in spleen and thymus of Dex group were significantly increased (P<0.01); compared with Dex group, the mRNA expressions of ASK1, JNK and P38 in spleen and thymus of 0.50 and 1.00 mg/kg BA groups were significantly decreased (P<0.05 or P<0.01). 3) Compared with NC group, the protein expressions of JNK and P38 in spleen of Dex group were significantly increased (P<0.01); compared with Dex group, the ASK1 protein expressions in spleen of 0.50 mg/kg BA group was significantly decreased (P<0.05), the protein expressions of JNK and P38 in spleen of 0.25, 0.50 and 1.00 mg/kg BA groups were significantly decreased (P<0.05 or P<0.01). In conclusion, BA pretreatment can increase T-AOC, ability of inhibiting hydroxyl radical and POD activity in liver, spleen and thymus of mice induced by Dex, decrease the gene expressions of ASK1, JNK and P38 in spleen and thymus and the protein expressions of ASK1, JNK and P38 in spleen of mice induced by Dex. BA shows preventive protection of oxidative damage induced by Dex, and the protection is related to JNK-P38 MAPK signal pathway.