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Pancreatic cancer is one of the most aggressive cancers. The vast majority of patients are diagnosed with advanced, unresectable disease because of early invasive growth and metastatic spread. The aim of this study was to examine YB-1 expression in pancreatic cancer and determine its effects on cell invasion. YB-1 is overexpressed in pancreatic cancer cell lines and patient tissue samples. In patient tissues, high YB-1 levels correlated with perineural invasion. Silencing of YB-1 significantly reduced cell invasion with decreased expression of MMPs in vitro. Furthermore, we found that the expression of YB-1 was suppressed by miR-216a via direct binding to the YB-1 3’-untranslated region. MiR-216a and YB-1 expression levels were inversely correlated in pancreatic cancer cell lines. In addition, ectopic expression of miR-216a inhibited cell invasion in vitro. Taken together, our findings suggest that YB-1 may play an important role in mediating metastatic behaviour and that repression of YB-1 by miR-216a could have a promising therapeutic potential to inhibit tumor metastasis in pancreatic cancer.