Immune checkpoint blockade therapy, one of the most promising cancer immunotherapies, has shown remarkable clinical impact in multiple cancer types. Despite the recent success of immune checkpoint blockade therapy, however, the response rates in patients with cancer are limited (∼20%–40%). To improve the success of immune checkpoint blockade therapy, relevant preclinical animal models are essential for the development and testing of multiple combination approaches and strategies. Companion dogs naturally develop several types of cancer that in many respects resemble clinical cancer in human patients. Therefore, the canine studies of immuno-oncology drugs can generate knowledge that informs and prioritizes new immuno-oncology therapy in humans. The challenge has been, however, that immunotherapeutic antibodies targeting canine immune checkpoint molecules such as canine PD-L1 (cPD-L1) have not been commercially available. Here, we developed a new cPD-L1 antibody as an immuno-oncology drug and characterized its functional and biological properties in multiple assays. We also evaluated the therapeutic efficacy of cPD-L1 antibodies in our unique caninized PD-L1 mice. Together, thesein vitroandin vivodata, which include an initial safety profile in laboratory dogs, support development of this cPD-L1 antibody as an immune checkpoint inhibitor for studies in dogs with naturally occurring cancer for translational research. Our new therapeutic antibody and caninized PD-L1 mouse model will be essential translational research tools in raising the success rate of immunotherapy in both dogs and humans.Significance:Our cPD-L1 antibody and unique caninized mouse model will be critical research tools to improve the efficacy of immune checkpoint blockade therapy in both dogs and humans. Furthermore, these tools will open new perspectives for immunotherapy applications in cancer as well as other autoimmune diseases that could benefit a diverse and broader patient population.