The present study investigated the role of microRNA (miR)-138-5p in regulating carcinoma migration and sensitivity to chemotherapy in pancreatic cancer. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to assess the expression levels of miR-138-5p in pancreatic cancer cell lines and primary carcinoma tissues from human patients. A lentiviral vector, containing miR-138-5p mimics (1v-miR-138-m) or miR-138-5p inhibitor (1v-miR-138-i), was used to either upregulate or downregulate the expression levels of miR-138-5p in PANC-1 cells, respectively. The effects of miR-138-3p regulation on pancreatic cancer cell migration and sensitivity to chemotherapy were examined. The predicted targeting of miR-138-5p on vimentin (VIM) was assessed by western blotting in PANC-1 cells. VIM was subsequently downregulated using small interfering (si)RNA to determine its effect on miR-138-5p-modulated pancreatic cancer cell development. The expression levels of miR-138-5p were downregulated in pancreatic cancer cell lines and primary carcinoma tissues. In PANC-1 cells, lentivirus-mediated upregulation of miR-138-5p inhibited cancer cell migration and increased cell chemosensitivity to 5-fluorouracil (5-FU). By contrast, downregulation of miR-138-5p promoted cancer cell migration and decreased cell chemosensitivity to 5-FU. A luciferase assay revealed that VIM was a direct target of miR-138-5p. Western blotting demonstrated that VIM was downregulated upon the upregulation of miR-138-5p in PANC-1 cells. siRNA-mediated downregulation of VIM inhibited pancreatic cancer cell migration in the control and miR-138-5p downregulated PANC-1 cells. The present study demonstrated that miR-138-5p is important in regulating pancreatic cancer development, possibly through targeting VIM.