Understanding the mechanism of radioresistance could help develop strategies to improve therapeutic response of patients with PDAC. The gene is frequently mutated in pancreatic cancer. In this study, we investigated the role of deficiency in pancreatic cancer cells' response to radiotherapy. We downregulated SMAD4 expression with siRNA or shRNA and overexpressed SMAD4 in mutant pancreatic cancer cells followed by clonogenic survival assay to evaluate their effects on cell radioresistance. To study the mechanism of radioresistance, the effects of loss on reactive oxygen species (ROS) and autophagy were determined by flow cytometry and immunoblot analysis, respectively. Furthermore, we measured radioresistance by clonogenic survival assay after treatment with autophagy inhibitor (Chloroquine) and ROS inhibitor (N-acetyl-l-cysteine) in -depleted pancreatic cancer cells. Finally, the effects of on radioresistance were also confirmed in an orthotopic tumor model derived from -depleted Panc-1 cells. -depleted pancreatic cancer cells were more resistant to radiotherapy based on clonogenic survival assay. Overexpression of wild-type SMAD4 in -mutant cells rescued their radiosensitivity. Radioresistance mediated by depletion was associated with persistently higher levels of ROS and radiation-induced autophagy. Finally, depletion induced radioresistance in Panc-1-derived orthotopic tumor model ( = 0.038). More interestingly, we observed that the protein level of SMAD4 is inversely correlated with autophagy in orthotopic tumor tissue samples. Our results demonstrate that defective is responsible for radioresistance in pancreatic cancer through induction of ROS and increased level of radiation-induced autophagy. .