Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease and is usually resistant to chemotherapy. MicroRNA‑181b (miR-181b) has been reported to be associated with chemoresistance in various types of cancer. In this study, we investigated the effects of miR-181b on the chemosensitivity of PDAC cells to gemcitabine and the underlying molecular events. miR-181b mimics and inhibitors were synthesized for transient gene transfection in vitro. Lentivirus carrying miR-181b mimics were used to infect PDAC cells for nude mouse xenograft assays by implanting infected PDAC cells into recipient mice. Cell viability was determined by MTT assays, while gene expression was assessed using qRT-PCR, western blot analysis and enzyme-linked immunosorbent assay (ELISA). miR-181b targeting BCL-2 expression was assessed by a dual-luciferase activity assay. The data showed that miRNA-181b expression sensitized PDAC cells to gemcitabine treatment. Although gemcitabine-resistant PDAC cell sublines (SW1990/GR and CFPAC-1/GR) expressed higher levels of miRNA-181b, gemcitabine induced higher levels of apoptosis in PDAC cells transfected with miRNA-181b mimics. The nude mouse xenograft assay data showed that miR-181b transfection also sensitized the cells to gemcitabine treatment in vivo. Molecularly, bioinformatics data predicted that miR-181b was able to bind to BCL-2 mRNA 3'UTR. The dual luciferase activity assay revealed that miRNA-181b downregulated BCL-2 expression. The results from western blot analysis showed a reduced BCL-2 expression following miR-181b transfection but an enhanced caspase-3 activity in miRNA-181b mimic-transfected PDAC cells. This study demonstrates that miRNA-181b sensitizes PDAC cells to gemcitabine by targeting BCL-2.