Non-equilibrium atmospheric pressure plasma (NEAPP) has attracted attention in cancer therapy. We explored the indirect effect of NEAPP through plasma-activated medium (PAM) on pancreatic cancer cells in vitro and in vivo. In this study, four pancreatic cancer cell lines were used and the antitumor effects of PAM treatment were evaluated using a cell proliferation assay. To explore functional mechanisms, morphological change and caspase-3/7 activation in cells were also assessed. Furthermore, reactive oxygen species (ROS) generation in cells was examined and N-acetyl cysteine (NAC), an intracellular ROS scavenger, was tested. Finally, the antitumor effect of local injection of PAM was investigated in a mouse xenograft model. We found that PAM treatment had lethal effect on pancreatic cancer cells. Typical morphological findings suggestive of apoptosis such as vacuolization of cell membranes, small and round cells and aggregation of cell nuclei, were observed in the PAM treated cells. Caspase-3/7 activation was detected in accordance with the observed morphological changes. Additionally, ROS uptake was observed in all cell lines tested, while the antitumor effects of PAM were completely inhibited with NAC. In the mouse xenograft model, the calculated tumor volume on day 28 in the PAM treatment group was significantly smaller compared with the control group [28 ± 22 vs. 89 ± 38 (mm(3) ± SD), p=0.0031]. These results show that PAM treatment of pancreatic cancer might be a promising therapeutic strategy.