Abstract Mucosal-associated invariant T (MAIT) cells are MR1-restricted innate-like T cells that recognize non-peptide antigens including riboflavin derivates. Although in vitro–activated MAIT cells show antitumor activity, the in vivo role of MAIT cells in cancer is still unclear. Here, we have shown that MAIT cells have antitumor function in vivo when activated by a combination of the synthetic riboflavin synthesis pathway–derived antigen 5-OP-RU [5-(2-oxopropylideneamino)-6-D-ribitylaminouracil] and the Toll-like receptor 9 (TLR9) agonist CpG. Coadministration of 5-OP-RU and CpG induced strong systemic in vivo expansion and activation of MAIT cells with high CD69 expression, pronounced effector memory phenotype, and upregulated levels of effector molecules including IFNγ, granzyme B, and perforin. Activated and expanded MAITs induced a potent and broad antitumor immune response in murine models of liver metastasis and hepatocellular carcinoma, lung metastasis, and subcutaneous tumors in two different mouse strains. Such tumor inhibition was absent in MAIT-deficient Mr1−/− mice. CRISPR/Cas9-mediated MR1 knockout in tumor cells did not affect efficacy of this MAIT-directed immunotherapy, pointing toward an indirect mechanism of action. Our findings suggest that MAIT cells are an attractive target for cancer immunotherapy. See related Spotlight by Lantz, p. 996.