Monoclonal antibodies against programmed cell death protein-1 (PD-1) and its ligand PD-L1 are immune checkpoint inhibitors (ICIs) that have been approved for the treatment of many human cancers. However, the response to these ICIs was limited in patients with ovarian cancer. Studies have indicated that the response to PD-1–PD-L1 blockade might be correlated with the PD-L1 expression level in cancer cells. Mechanistic pathways that control PD-L1 expression are yet to be elucidated and targeting these pathways can improve the efficacy of PD-1–PD-L1 blockade in patients with ovarian cancer. Our results revealed that NPM/B23 regulated PD-L1 expression in ovarian cancer cells. Mechanistically, NPM/B23 negatively regulates PD-L1 expression through NF-κB/p65. Moreover, in the immunocompetent mouse model, the NPM/B23 inhibitor/modulator sensitized the cancer cells to the anti-PD-1 antibody by regulating PD-L1 expression. Compared with treatment with the NPM/B23 inhibitor/modulator or the anti-PD-1 antibody alone, their combined use reduced tumorigenesis and increased CD8+ T-cell expansion, thus contributing to prolong the survival of MOSEC/Luc-bearing mice in vivo. In conclusion, targeting NPM/B23 is a novel therapeutic approach to sensitize cancer cells to immune therapy and reduce tumorigenesis.