Background <p>Pancreatic cancer is a common malignant tumor. We focused on exploring the function of miR-641 in stem cell characteristics for pancreatic cancer cells.</p> Method <p>MiR-641 expression was analyzed based on The Cancer Genome Atlas (TCGA) pancreatic cancer database and clinical samples. The miR-641 knockdown within pancreatic cancer was performed by cell transfection. CCK8, transwell, and flow cytometry were performed for analyzing cell growth, invasion as well as stem-cell-like features separately. Meanwhile, this study carried out the dual luciferase reporter gene assay. <i>In viv</i>o xenograft tumor assay was also performed.</p> Results <p>MiR-641 expression increased in clinical pancreatic cancer tissues and cells compared with normal cells. MiR-641 predicted poor survival rate of pancreatic cancer patients. MiR-641 down-regulation inhibited pancreatic cancer cell proliferation, clone forming ability, invasion and migration. Down-regulation of miR-641 inhibited the capability of sphere-forming, reduced CD133 + and CD44 + cell quantities, and suppressed CD133, CD44, and Oct4 expression in Down-regulation of miR-641 cells. MiR-641 targeted TMEFF2/MEK/PI3K for promoting pancreatic cancer cells’ stem-cell-like characteristics. MiR-641 also promoted tumor growth in vivo.</p> Conclusion <p>MiR-641 acts as an oncogene that promotes pancreatic cancer cell growth, invasion as well as stem-cell-like features, which is realized by regulating the expression of TMEFF2.</p>