Pathogens and cancer cells employ the programmed cell death-Ligand 1 (PD-L1)/ programmed cell death-1 (PD-1) signaling pathway to inhibit the immune response. Hence, blockade of PD-L1/PD-1 recognition through monoclonal antibodies enhances the immune response. Antibodies that block PD-L1 and PD-1 binding have been used for the prevention and therapy of human pathogenic diseases, but have not yet been evaluated for the treatment of infectious diseases of livestock. In the present study, a recombinant vaccine named PROF-PDL1E, was designed comprising the Babesia microti-derived vaccine candidate profilin and the host PD-L1 protein, and its effect on immunization against murine B. microti infection was evaluated. PD-L1-specific antibodies generated after vaccination blocked PD-L1 and PD-1 binding as shown by in vitro assays. PROF-PDL1E reduced the burden of B. microti in a mouse model and decreased PD-1 expression in T cells. Furthermore, no tissue damage could be observed after PROF-PDL1E vaccination as verified by hematoxylin and eosin tissue staining of essential organs. In conclusion, vaccines targeting immune checkpoints seem to be a promising strategy for anti-Babesiavaccine development.