Glioma is a CNS tumor with few therapeutic options. Recently, host microbiota has been involved in the immune modulation of different tumors, but no data are available on the possible effects of the gut-immune axis on brain tumors. Here, we investigated the effect of gut microbiota alteration in a syngeneic (GL261) mouse model of glioma, treating mice with two antibiotics (ABX) and evaluating the effects on tumor growth, microbe composition, natural killer (NK) cells and microglia phenotype. We report that ABX treatment (i) altered the intestinal microbiota at family level, (ii) reduced cytotoxic NK cell subsets, and (iii) altered the expression of inflammatory and homeostatic proteins in microglia. All these findings could contribute to the increased growth of intracranial glioma that was observed after ABX treatment. These results demonstrate that chronic ABX administration alters microbiota composition and contributes to modulate brain immune state paving the way to glioma growth.