We report optimization by rational design of JMPDP-027, a potent cyclic peptide that interferes with the PD-1/PD-L1 protein–protein interaction. JMPDP-027 shows a potent restoring ability towards T-cells with an EC₅₀ of 5.9 nM that is comparable to that of the anti-PD-1 monoclonal antibody pembrolizumab. In addition, JMPDP-027 shows not only high resistance to enzymatic hydrolysis in human serum but also no observable toxicity and potent in vivo anticancer activity comparable to that of the mouse PD-L1 antibody in a colon carcinoma (CT26) model. Cyclic peptide antagonists of this sort may provide novel drug candidates for cancer immunotherapy.