Pancreatic ductal adenocarcinoma (PDA) is a highly aggressive and often lethal malignant tumor. Several studies have shown that epithelial-mesenchymal transition (EMT) is frequently observed in clinical samples of PDA and is related to high metastatic rates and poor outcomes. To identify candidate molecules regulating EMT in PDA, we previously used cDNA microarray analysis and identified integrin β4 (ITGB4) as one of the genes upregulated in high-EMT xenografts derived from PDA patients. The aim of the current study was to clarify the clinicopathological and functional significance of ITGB4 overexpression in PDA. ITGB4 upregulation in high-EMT xenografts was confirmed by immunohistochemistry. Immunohistochemical analyses of 134 surgically resected PDA cases revealed intratumoral heterogeneity with respect to ITGB4 expression and showed that cancer cells undergoing EMT often display strong diffuse ITGB4 expression. High levels of ITGB4 expression were significantly correlated with the hallmarks of EMT (solitary cell infiltration, reduced E-cadherin expression, and increased vimentin expression), with high tumor grade, and with the presence of lymph node metastasis, and showed an independent prognostic effect. Immunocytochemical analyses of PDA cell lines revealed that localization of ITGB4 changed from regions of cell-cell contact to diffuse cytoplasm and cell edges with occasional localization in filopodia during EMT. Knockdown of ITGB4 reduced the migratory and invasive ability of PDA cells. Overexpression of ITGB4 promoted cell scattering and cell motility in combination with downregulation of E-cadherin and upregulation of vimentin expression. In conclusion, we elucidated the prognostic and clinicopathological significance of ITGB4 overexpression in PDA and also the potential role for ITGB4 in the regulation of cancer invasion and EMT.