Upregulation of programmed death 1 ligand 1 (PD-L1) in cancer cells and its ligation to PD-1 on T cells facilitates cancer cell escape from immune surveillance. Therapies with PD-1 or PD-L1 antibodies have resulted in marked clinical responses in various cancer types. Hence, modulators that inhibit PD-L1 expression in cancer cells may serve as a novel strategy by which to enhance host immune responses. In the present study, we investigated the effects of miR-375 on PD-L1 expression in head and neck squamous cell carcinoma (HNSCC) cells by qRT-PCR and western blot analyses. We confirmed that miR-375 inhibited IFN-γ-induced PD-L1 surface expression in HNSCC cells, and we observed that Janus kinase 2 (JAK2) is a bona fide target of miR-375 and further activated signal transducer and activator of transcription 1 (STAT1). Additionally, miR-375-mediated inhibition of PD-L1 expression was dependent on the JAK2/STAT1 pathway. Therefore, by attenuating PD-1/PD-L1 signaling, miR-375 may also serve as a modulator to increase the cell immune responses to HNSCC.