Immunotherapy, particularly with regard to cancer treatment, is currently an area of focus in research, but little is known about the function of immune inhibitor molecules in veterinary medicine. In this study, monoclonal antibodies against 2 immune inhibitory receptors (ie, canine programmed death-1 [cPD-1], canine programmed death ligand-1 [cPD-L1]) were identified. These antibodies bound to cells overexpressing cPD-1 and cPD-L1, effectively blocking binding between cPD-1 and cPD-L1. Each antibody was found to have a different blocking ability and a specific functional blockade. These newly generated antibodies may provide a novel approach to treatment of dogs with immunosuppressive cancers.