Immunotherapy is rapidly emerging as a cancer treatment with high potential. Recent clinical trials with anti-CTLA-4 and anti–PD-1/PD-L1 antibodies (mAbs) suggest that targeting multiple immunosuppressive pathways may significantly improve patient survival. The generation of adenosine by CD73 also suppresses antitumor immune responses through the activation of A_2A receptors on T cells and natural killer (NK) cells. We sought to determine whether blockade of A_2A receptors could enhance the efficacy of anti–PD-1 mAb. The expression of CD73 by tumor cells limited the efficacy of anti–PD-1 mAb in two tumor models, and this was alleviated with concomitant treatment with an A_2A adenosine receptor antagonist. The blockade of PD-1 enhanced A_2A receptor expression on tumor-infiltrating CD8⁺ T cells, making them more susceptible to A_2A-mediated suppression. Thus, dual blockade of PD-1 and A_2A significantly enhanced the expression of IFNγ and Granzyme B by tumor-infiltrating CD8⁺ T cells and, accordingly, increased growth inhibition of CD73⁺ tumors and survival of mice. The results of our study indicate that CD73 expression may constitute a potential biomarker for the efficacy of anti–PD-1 mAb in patients with cancer and that the efficacy of anti–PD-1 mAb can be significantly enhanced by A_2A antagonists. We have therefore revealed a potentially novel biomarker for the efficacy of anti–PD-1 that warrants further investigation in patients. Because our studies used SYN-115, a drug that has already undergone phase IIb testing in Parkinson disease, our findings have immediate translational relevance for patients with cancer. Cancer Immunol Res; 3(5); 506–17. ©2015 AACR.