α-Difluoromethylornithine (DFMO) is currently used in chemopreventive regimens primarily for its conventional direct anticarcinogenesic activity. However, little is known about the effect of ornithine decarboxylase (ODC) inhibition by DFMO on antitumor immune responses. We showed in this study that pharmacologic blockade of ODC by DFMO inhibited tumor growth in intact immunocompetent mice, but abrogated in the immunodeficient Rag1−/− mice, suggesting that antitumor effect of DFMO is dependent on the induction of adaptive antitumor T cell immune responses. Depletion of CD8+ T cells impeded the tumor-inhibiting advantage of DFMO. Moreover, DFMO treatment enhanced antitumor CD8+ T cell infiltration and IFN-γ production and augmented the efficacy of adoptive T cell therapy. Importantly, DFMO impaired Gr1+ CD11b+ myeloid-derived suppressor cells (MDSCs) suppressive activity through at least two mechanisms, including reducing arginase expression and activity and inhibiting the CD39/CD73-mediated pathway. MDSCs were one primary cellular target of DFMO as indicated by both adoptive transfer and MDSC-depletion analyses. Our findings establish a new role of ODC inhibition by DFMO as a viable and effective immunological adjunct in effective cancer treatment, thereby adding to the growing list of chemoimmunotherapeutic applications of these agents.