Chemoresistance is an obstacle of improving pancreatic cancer (PC) prognosis. However, the biological function of ISG15 in PC and whether it correlates with the resistance to chemotherapy are still unknown. Here, we aimed to reveal the clinical significance of ISG15 in PC and its regulatory mechanism in cancer progression and resistance to therapy. The level of ISG15, a protein involved in post-translational modifications, is elevated in PC tissues. Clinically, higher ISG15 expression correlates with higher PC grades, stronger resistance to treatment and poorer prognosis. Moreover, ISG15 promotes the proliferation, migration, invasion, colony formation of PC cells and resistance to Gemcitabine, a classic chemotherapeutics for PC, both in vitro and in vivo. ISG15 promotes progression and resistance to therapy in PC cells by binding to ATG7, reducing its degradation, and thereby leading to enhanced autophagy in PC cells. ISG15 may be used as both a potential diagnosis marker and sensitizer for chemotherapeutics such as Gemcitabine during PC intervention.