Gamma irradiation-based mutant creation is one of the most important methods for rice plant mutagenesis breeding and molecular biology research. Although median lethal dose irradiation severely damages rice seedlings, applying brassinolide (BR) can increase the survival rate of irradiated seedlings. In this study, we investigated the effects of soaking seeds in solutions containing different BR concentrations (0.001, 0.01, 0.1, 1.0, and 5.0 μmol/L) and then spraying the resulting seedlings twice with 0.1 μmol/L BR. The combined BR treatments markedly decreased the superoxide anion (O2•−), hydrogen peroxide (H2O2), and malondialdehyde contents but increased the chlorophyll content. An appropriate BR treatment of gamma-irradiated samples substantially increased the activities of the antioxidant enzymes superoxide dismutase, peroxidase, and ascorbate peroxidase as well as the proline, ascorbic acid, and glutathione contents in rice seedling shoots. The BR treatment also promoted the growth of seedlings derived from irradiated seeds and increased the shoot and root fresh and dry weights. Most notably, soaking seeds in 0.01 or 0.1 μmol/L BR solutions and then spraying seedlings twice with 0.1 μmol/L BR significantly increased the final seedling survival rate and decreased mutant loss. The study results suggest that exogenous BR treatments can protect rice seedlings from gamma irradiation stress by enhancing antioxidant metabolism.