Programmed cell death protein 1 (PD-1) blockade is a promising therapeutic strategy against prostate cancer. Nitroxoline has been found to have effective anticancer properties in several cancer types. We investigated the efficacy of a combination therapy involving nitroxoline and PD-1 blockade in a prostate cancer mouse model. In our in vitro analysis, we found that nitroxoline inhibited the viability and proliferation of the mouse prostate cancer cell line RM9-Luc-PSA. Additionally, nitroxoline downregulated the expressions of phospho-PI3 kinase, phospho-Akt (Thr308), phospho-Akt (Ser473), phospho-GSK-3β, Bcl-2, and Bcl-xL. Nitroxoline also downregulated programmed death-ligand 1 (PD-L1) expression levels in prostate cancer cell line and tumor tissue. In our murine prostate cancer orthotopic model, nitroxoline plus PD-1 blockade synergistically suppressed tumor growth when compared with nitroxoline or PD-1 blockade alone, leading to reductions in tumor weight, bioluminescence tumor signals, and serum prostate-specific antigen levels. Furthermore, fluorescence-activated cell sorting analysis showed that the combination strategy significantly enhanced antitumor immunity by increasing CD44+CD62L+CD8+ memory T cell numbers and reducing myeloid-derived suppressor cell numbers in peripheral blood. In conclusion, our findings suggest that nitroxoline plus PD-1 blockade may be a promising treatment strategy in patients with prostate cancer.