Pancreatic cancer is characterized by occult onset, low early diagnosis rate, rapid progress, and poor prognosis. Due to the low response rate and low programmed cell death ligand 1 (PD-L1) expression in pancreatic cancer, the therapeutic application of PL-L1 inhibitors in pancreatic cancer is greatly limited. In vitro studies showed that the expression of PD-L1 increased in pancreatic cancer cells stimulated by fluorouracil (5-FU). We aim to explore the combined effect of 5-FU and anti-PD-L1 antibodies and to provide a reference for the clinical application of PD-L1 antibodies in pancreatic cancer. In the current study, male BALB/c mice were adopted to construct a tumor-bearing model of pancreatic cancer cells. 5-FU and anti-mouse PD-L1 antibodies were combined and administered to evaluate their synergistic effects. The enhancing immune cytotoxicity effect of 5-FU sensitizing the anti-PD-L1 antibody in vivo and in vitro was analyzed by immunohistochemistry and western blot assays. Results showed that 5-FU and anti-PD-L1 antibody combination increased the expression of PD-L1 and IFN-γ, and infiltration of CD8