Pancreatic cancer (PC) is a lethal solid malignancy with resistance to traditional chemotherapy. Recently, considerable studies have demonstrated the ubiquitous antitumor properties of gene therapy mediated by the oncolytic vaccinia virus. The second mitochondrial‑derived activator of caspase (Smac) has been identified as an innovative tumor suppressor that augments the chemosensitivity of cancer cells. However, the therapeutic value of oncolytic vaccinia virus (oVV)‑mediated Smac gene transfer in pancreatic cancer is yet to be elucidated. In the present study, oncolytic vaccinia virus expressing Smac (second mitochondrial‑derived activator of caspase) (oVV‑Smac) was used to examine its beneficial value when used alone or with gemcitabine in pancreatic cancer in vitro and in vivo. The expression of Smac was evaluated by western blot analysis and quantitative polymerase chain reaction, oVV‑Smac cytotoxicity by MTT assay, and apoptosis by flow cytometry and western blot analysis. Furthermore, the inhibitory effect of oVV‑Smac combined with gemcitabine was also evaluated. The results indicated that oVV‑Smac achieved high levels of Smac, greater cytotoxicity, and potentiated apoptosis. Moreover, co‑treatment with oVV‑Smac and gemcitabine resulted in a synergistic effect in vitro and in vivo. Therefore, our findings advance oVV‑Smac as a potential therapeutic candidate in pancreatic cancer and indicated the synergistic effects of co‑treatment with oVV‑Smac and gemcitabine.