Castration-resistant prostate cancer (CRPC) is difficult to treat in current clinical practice. Hypoxia is an important feature of the CRPC microenvironment and is closely associated with the progress of CRPC invasion. However, no research has been performed on the immune escape of CRPC from NK cells. The present study focused on this subject. Firstly, when the CRPC cell lines C4-2 and CWR22Rv1 were induced by hypoxia, the expression of the UL16 binding protein (ULBP) ligand family of natural killer (NK) group 2D (NKG2D; ULBP-1, ULBP-2 and ULBP-3) and MHC class I chain-related proteins A and B (MICA/MICB) decreased. NKG2D is the main activating receptor of NK cells. Tumor cells were then co-cultured with NK cells to conduct NK cell-mediated cytotoxicity experiments, which revealed the decreased immune cytolytic activity of NK cells on hypoxia-induced CRPC cells. In exploring the mechanism behind this observation, an increase in programmed death-ligand 1 (PD-L1) expression in CRPC cells induced by hypoxia was observed, while the addition of PD-L1 antibody effectively reversed the expression of NKG2D ligand and enhanced the cytotoxic effect of NK cells on CRPC cells. In the process of exploring the upstream regulatory factors of PD-L1, inhibition of the Janus kinase (JAK)(1,2)/signal transducer and activator of transcription 3 (Stat3) signaling pathway decreased the expression of PD-L1 in CRPC cells. Finally, it was observed that combined inhibition of JAK(1,2)/PD-L1 or Stat3/PD-L1 was more effective than inhibition of a single pathway in enhancing the immune cytolytic activity of NK cells. Taking these results together, it is thought that combined inhibition of the JAK(1,2)/PD-L1 and Stat3/PD-L1 signaling pathways may enhance the immune cytolytic activity of NK cells toward hypoxia-induced CRPC cells, which is expected to provide novel ideas and targets for the immunotherapy of CRPC.