Autophagy has recently emerged as a significant mechanism in cancer treatment. Although gemcitabine and/or ionizing radiation are important modalities in the treatment of pancreatic cancer, the contribution of autophagy in such treatment has not been fully elucidated. This study investigated the role of autophagy in the treatment of pancreatic cancer with gemcitabine and ionizing radiation. To evaluate the effect of gemcitabine and/or ionizing radiation on autophagy, several human pancreatic cancer cell lines were used. The treatment of pancreatic cancer cell cultures in vitro and in vivo with gemcitabine and ionizing radiation resulted in synergistic cytotoxicity. After treatment with gemcitabine, the autophagy-related protein light chain 3-II (LC3-II) was upregulated. When gemcitabine was combined with ionizing radiation treatment, LC3-II upregulation was enhanced. In addition, electron microscopy of pancreatic cancer cells treated with gemcitabine and/or ionizing radiation detected the induction of autophagy. The blockage of autophagy by 3-methyladenine indicated that autophagy contributed to cell death after gemcitabine treatment and enhanced its cytotoxicity. The inhibitory effect and immune reactivity of the autophagy-related proteins LC3 and beclin-1 were the strongest after the combination treatment. In conclusion, these results suggest that autophagy can be activated by gemcitabine and/or ionizing radiation in the treatment of pancreatic cancer cells and that activated autophagy plays a role in cancer suppression. These findings may have important implications for future therapeutic strategies using gemcitabine and ionizing radiation against pancreatic cancer.