Several species of intestinal bacteria have been associated with enhanced efficacy of checkpoint blockade immunotherapy, but the underlying mechanisms by which the microbiome enhances antitumor immunity are unclear. In this study, we isolated three bacterial species- , , and species-that significantly enhanced efficacy of immune checkpoint inhibitors in four mouse models of cancer. We found that intestinal modulated enhanced immunotherapy response through production of the metabolite inosine. Decreased gut barrier function induced by immunotherapy increased systemic translocation of inosine and activated antitumor T cells. The effect of inosine was dependent on T cell expression of the adenosine A receptor and required costimulation. Collectively, our study identifies a previously unknown microbial metabolite immune pathway activated by immunotherapy that may be exploited to develop microbial-based adjuvant therapies.