Proton minibeam radiation therapy (pMBRT) is a novel radiation therapy approach that exploits the synergies of proton therapy with the gain in normal tissue preservation observed upon irradiation with narrow, spatially fractionated, beams. The net gain in normal tissue sparing that has been shown by pMBRT may lead to the efficient treatment of very radioresistant tumors, which are currently mostly treated palliatively. The aim of this study was to perform an evaluation of the tumor effectiveness of proton minibeam radiation therapy for the treatment of RG2 glioma-bearing rats.Two groups (n = 9) of RG2 glioma-bearing rats were irradiated with either standard proton therapy or with pMBRT, with a dose prescription of 25 Gy in 1 fraction. The animals were followed up for a maximum of 6 months. At the end of the study, histopathological studies were performed to assess both the tumor presence and the possible side effects.Tumor control was achieved in the 2 irradiated series, with superior survival in the pMBRT group compared with the standard proton therapy group. Long-term (>170 days) survival rates of 22% and 67% were obtained in the standard proton therapy and pMBRT groups, respectively. No tumor was observed in the histopathological analysis. Although animals with long-term survival in the standard radiation therapy exhibit substantial brain damage, including marked radionecrosis, less severe toxicity was observed in the pMBRT group.pMBRT offers a significant increase in the therapeutic index of brain tumors: The majority of the glioma-bearing rats (67%) survived 6 months with less severe side effects.