Gemcitabine is widely used as an anticancer chemotherapy drug for a variety of solid tumors, and it has become the standard treatment option for locally advanced and metastatic pancreatic cancer. However, pancreatic cancer cells develop resistance to gemcitabine after a few weeks of treatment, resulting in poor therapeutic effects. Isocorydine (ICD) is a typical natural aporphine alkaloid, and ICD and its derivatives inhibit the proliferation of many types of cancer cells in vitro. In this study, ICD was found to synergistically inhibit cell viability with gemcitabine in pancreatic cancer cells. A microarray analysis showed that ICD can inhibit the upregulation of STAT3 and EMT in pancreatic cancer cells induced by gemcitabine. STAT3 is closely related to tumor EMT, migration and invasion. After knocking down the expression of STAT3 in pancreatic cancer cells, the combination index (CI) of ICD and gemcitabine decreased. ICD can reverse the increase in the expression of EMT-related transcription factors and proteins caused by gemcitabine, thereby inhibiting the enhanced cell migration and invasion ability caused by gemcitabine. Finally, the synergistic treatment effect of the combination treatment of ICD and gemcitabine in pancreatic cancer cells was confirmed in established xenograft models.