Pancreatic cancer is a highly aggressive malignancy with a poor prognosis, mainly due to late diagnosis and early metastatic spread. The underlying mechanisms of pancreatic cancer metastasis, particularly the role of hepatic stellate cell (HSC) activation, are not fully understood. This study tried to find potential biomarkers for pancreatic cancer progression and prognosis by analyzing extracellular vesicle (EV)-associated miRNA profiles in plasma from pancreatic cancer patients and non-cancer controls. Functional assays, including transfection, western blotting, immunofluorescence, and enzyme-linked immunosorbent assay (ELISA), were used to assess the ability of the identified miRNA to activate HSCs and promote cancer progression. Our findings revealed that miR-25-3p was significantly upregulated in EVs derived from pancreatic cancer patients, correlating with increased metastasis and worse survival outcomes. EV-associated miR-25-3p from metastatic pancreatic cancer cells activated HSCs by regulating the expression of Krüppel-like factor 4 (KLF4). Additionally, activated HSCs secreted vascular endothelial growth factor (VEGF), further driving pancreatic cancer metastasis and progression. These results suggest that miR-25-3p could serve as a novel biomarker for pancreatic cancer progression and a potential therapeutic target to improve patient outcomes.