Pancreatic cancer is one of the most lethal malignancies, partly due to its high resistance to conventional chemotherapy. The aim of this study is to investigate the association between chemoresistance and trefoil factor family 1 (TFF1), a tumor-suppressive protein in pancreatic carcinogenesis. To investigate the role of TFF1 in human and mice, specimens of human pancreatic cancer and genetically engineered mouse model of pancreatic cancer (KPC/TFF1KO; Pdx1-Cre/ LSL-KRASG12D/ LSL-p53R172H/ TFF1-/-) were analysed. The expression of TFF1 in cancer cells was associated with better survival of the patients who underwent chemotherapy, and the deficiency of TFF1 increased EMT of cancer cells and deteriorated the benefit of gemcitabine in mice. To explore the efficacy of TFF1 treatment, recombinant and chemically synthesized TFF1 were administered to pancreatic cancer cell lines and mouse models. TFF1 inhibited gemcitabine-induced EMT, Wnt pathway activation and cancer stemness, eventually increased apoptosis of pancreatic cancer cells by gemcitabine. Combined treatment of gemcitabine and TFF1 arrested tumor growth and resulted in the better survival of mice. These results indicate that TFF1 can contribute to establishing a novel strategy to treat pancreatic cancer patients by enhancing chemosensitivity.