Pancreatic cancer is one of the most malignant tumors with high mortality and poor prognosis even with the aggressive conventional therapies. Biotherapy based on the understanding of tumorigenesis mechanism is ongoing to improve the outcomes of cancer patients. We sought here to evaluate the therapeutic potential of a proapoptotic gene, PUMA, in pancreatic cancer. We found that PUMA was differently expressed in a series of pancreatic ductal adenocarcinoma cancer cell lines, and adenovirus-mediated expression of PUMA (Ad-PUMA) in these cells resulted in massive apoptosis. PUMA was more potent than p53 in suppressing growth of cancer cells. RT-PCR and Western Blot revealed that exogenous PUMA was expressed 6 h after Ad-PUMA infection. Furthermore, we assessed the efficacy of Ad-PUMA combining anticancer drugs (5-fluorouracil, cisplatin, gemcitabine hydrochloride, respectively) in these pancreatic cancer cell lines. Data revealed that PUMA significantly sensitized pancreatic carcinoma cell lines to chemotherapeutics, which may be resulted from abundant apoptosis induction. In nude mice with PANC-1 xenografts, Ad-PUMA treatment significantly inhibited the tumor growth. These results suggest that PUMA is a potent molecular tool in suppressing tumor growth sensitizing pancreatic carcinoma cells to chemical drugs. PUMA plays roles in negatively regulating cancer cell growth and may be a promising tool for cancer biotherapy, with or without combination with chemotherapeutic agents.