Abstract Radio-resistance is a growing concern in treating patients with pancreatic cancer (PC). Here we investigated the role of miR-590-5p in the radio-resistance of PC cells. We developed radioresistant PC cell lines and followed by microarray analysis and levels of miRs compared to parental cell lines. PC cells were transfected using either miR mimics or inhibitors followed by clonogenic survival assays. We also studied the effect of miR-590-5p on autophagy using electron microscopy and immunoblot analysis. In addition, the luciferase assay was used to identify potential targets. The radio-resistant PC cells exhibited decreased expression of miR-590-5p, with elevated autophagy against the parental cells. The over-expression of miR-590-5p inhibited radiation-mediated autophagy, while inhibitors induced autophagy in PC cells. The up-regulation of miR-590-5p enhanced the radio-sensitivity of PC cells. We confirmed ATG-3 as a target of miR-590-5p, whose levels were unregulated in radio-resistant cells. We also found that levels of ATG-3 were associated with autophagy. Expression of miR-590-5p inhibited radiation-mediated autophagy and enhanced the radio-sensitivity of PC cells.