Resistance to first-line chemotherapeutic drugs such as gemcitabine contributes to the poor prognosis of patients with pancreatic cancer. MicroRNAs (miRNA) regulate chemoresistance in pancreatic cancer. By analyzing the miRNA sequencing dataset of pancreatic cancer from The Cancer Genome Atlas, it was demonstrated that miR-374b-5p expression was dramatically reduced in pancreatic cancer tissues compared with adjacent normal tissues, as well as decreased in chemoresistant compared with chemosensitive pancreatic carcinoma tissues. The decreased expression of miR-374-5p was associated with poor overall and progression-free survival in patients with pancreatic cancer. Furthermore, increased expression of miR-374b-5p abrogated, while the silencing miR-374b-5p increased the chemoresistance of pancreatic cancer cells to gemcitabine in vitro. Importantly, the upregulation of miR-374b-5p ameliorated the chemoresistance of pancreatic cancer cells to gemcitabine in vivo. It was also demonstrated that miR-374b-5p targeted several anti-apoptotic proteins, including B-cell lymphoma 2, Baculoviral IAP Repeat Containing 3 and X-linked inhibitor of apoptosis in pancreatic cancer cells, which further attenuated chemo-resistance in pancreatic cancer. Therefore, the results of the current study indicate that miR-374b-5p serves as a potential diagnostic marker. It also suggests that miR-374b-5p sensitizes cells to chemotherapy and may be used in combination with chemotherapeutic agents such as gemcitabine to treat patients with pancreatic cancer.