This study was focused on the evaluation of radical scavenging effect and the protective activity against oxidative stress of the extract and fractions from Petasites japonicus. P. japonicus was extracted with methanol and then fractionated into 4 fractions [n-butanol, ethyl acetate (EtOAc), methylene chloride, and n-hexane]. The extract and fractions showed strong 1,1- diphenyl-2-picrylhydrazyl radical scavenging activity. Among all the fractions, particularly, the EtOAc fraction showed the strongest effect with the IC50 value of 0.02 μg/ml. In addition, the fractions also showed strong hydroxyl radical scavenging activity and nitric oxide scavenging activity as well. Furthermore, cell viability generated by the P. japonicus extract and 4 fractions were examined under C6 glial cellular model. The C6 glial cells showed high generation of reactive oxygen species (ROS) and decrease in cell viability by the treatment generator of hydrogen peroxide. However, the production of ROS formation was decreased by the treatment of the fractions of P. japonicus and also founded that the EtOAc fraction led to significant increase in the cell viability at concentration 100 μg/ml. Results from this work indicated that P. japonicus showed protective effects against oxidative stress and its EtOAc fraction may be served as a useful natural antioxidant.