As a conventional immunosuppressive drug, cyclophosphamide (CYP) exhibits strong hepatotoxicity in clinical applications. Betulinic acid (BA) is a natural triterpenoid that protects against liver damage. However, the underlying mechanism has not yet been elucidated. The purpose of this study was to evaluate the ameliorative effects of BA on CYP-induced hepatotoxicity and further clarify the underlying mechanism. BA pretreatment mitigated CYP-induced liver oxidative damage by alleviating histopathological lesions, reducing reactive oxygen species (ROS) accumulation, and restoring the mRNA expression of antioxidant enzymes (Cu-Sod, Mn-Sod, Cat, and Gsh-Px). BA treatment also suppressed CYP-induced oxidative stress by activating the NRF2 pathway and inhibiting the MAPK signaling pathway. Moreover, BA attenuated CYP-triggered hepatic apoptosis by suppressing excessive mitochondrial fission, boosting mitochondrial fusion, and ameliorating pro-apoptotic protein expression (CASP9 and the ratio of BCL-2/BAX) by blocking the oxidative stress-activated mitochondrial apoptotic pathway. Furthermore, PD98059 (an inhibitor of ERK) and/or BA abated CYP-provoked hepatotoxicity by inhibiting the ERK-MAPK and mitochondrial apoptotic pathways, implying that deactivation of the ERK-mediated mitochondrial apoptotic pathway contributed to the hepatoprotective efficacy of BA against CYP-induced oxidative stress. Therefore, BA could be used as a complementary medicine in patients undergoing CYP treatment owing to its hepatoprotective effects.