Microbeam radiation therapy (MRT), a preclinical form of radiosurgery, uses spatially fractionated micrometre-wide synchrotron-generated X-ray beams. As MRT alone is predominantly palliative for animal tumors, the effects of the combination of MRT and a newly synthesized chemotherapeutic agent JAI-51 on 9L gliosarcomas have been evaluated. Fourteen days (D14) after implantation (D0), intracerebral 9LGS-bearing rats received either MRT, JAI-51 or both treatments. JAI-51, alone or immediately after MRT, was administered three times per week. Animals were kept up to ∼20 weeks after irradiation or sacrificed at D16 or D28 after treatment for cell cycle analysis. MRT plus JAI-51 increased significantly the lifespan compared with MRT alone (p = 0.0367). JAI-51 treatment alone had no effect on rat survival. MRT alone or associated with JAI-51 induced a cell cycle blockade in G2/M (p < 0.01) while the combined treatment also reduced the proportion of G0/G1 cells. At D28 after irradiation, MRT and MRT/JAI-51 had a smaller cell blockade effect in the G2/M phase owing to a significant increase in tumor cell death rate (<2c) and a proportional increase of endoreplicative cells (>8c). The combination of MRT and JAI-51 increases the survival of 9LGS-bearing rats by inducing endoreduplication of DNA and tumor cell death; further, it slowed the onset of tumor growth resumption two weeks after treatment.