The present study aimed to investigate the effect of the negative costimulatory molecule programmed death-ligand 1 (PD-L1) on immunotherapy with OK-432, following transurethral resection of bladder tumors in non-muscle invasive bladder cancer (NMIBC), and to elucidate the underlying mechanism. PD-L1 was detected by immunohistochemical staining in tumor specimens from 55 cases of NMIBC following postoperative immunotherapy with OK-432. The PD-L1 mRNA and protein expression levels were measured in the bladder cancer T24 cell line and the human uroepithelial SV-HUC-1 cell line, following treatment with interleukin (IL)-2, interferon (IFN)-alpha and IFN-gamma, by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis, respectively. PD-L1 was widely expressed in the NMIBC tumors, with 56.4% (31/55) of specimens exhibiting positive staining. When compared with PD-L1-negative patients, PD-L1-positive patients exhibited significantly increased recurrence [48.4% (15/31) vs. 16.7% (4/24)] and progression [16.1% (5/31) vs. 4.2% (1/24)] rates (P<0.05). RT-qPCR and western blotting demonstrated that cytokines IL-2, IFN-alpha and IFN-gamma markedly upregulated PD-L1 mRNA expression rates and protein levels in bladder cancer T24 cells (P<0.05), but had no significant effect in non-tumor SV-HUC-1 cells. In conclusion, PD-L1 expression was negatively-associated with the efficacy of OK-432 intravesical immunotherapy in patients with NMIBC. The results indicated that the involved mechanism occurred via upregulation of PD-L1 by immune cytokines, which in turn suppressed the antitumor effectiveness of the immune system, thereby promoting tumor recurrence and progression.