Mitogen-activated protein kinase signal transduction pathway was isolated as a potential factor related to cancer cell dissociation in dissociated (PC-1.0 and AsPC-1) and non-dissociated (PC-1 and Capan-2) pancreatic cancer cells in our previous works. On the other hand, changes of structure and function of tight junction (TJ) are reported to be correlated with carcinogenesis and tumor development. In this study, the translocation of TJ protein Zonula occludens-1 (ZO-1) and the activation of epidermal growth factor receptor (EGFR) were examined to demonstrate the involvement and correlation of TJ protein translocation and EGFR activation in the cell dissociation and subsequent invasion of pancreatic cancer. Immunocytochemistry, fluorescence intensity analysis and in vitro invasion assay were performed in dissociated and non-dissociated pancreatic cancer cells. The obvious translocation of cell-cell junction localized ZO-1 protein to the cytoplasm and nucleus, simultaneous activation of EGFR, as well as the dissociation of cell colonies of non-dissociated pancreatic cancer cells were induced by dissociation factor treatment. However, EGFR inhibitor, AG1478, treatment significantly induced the redistribution of ZO-1 protein to the sites of cell-cell junction and the cell aggregation, as well as simultaneous suppression of EGFR activation in both the dissociated and the non-dissociated pancreatic cancer cells. In addition, AG1478 treatment markedly enhanced the in vitro invasion of non-dissociated pancreatic cancer cells. Translocation of TJ protein ZO-1 is closely involved in the induction of invasion through EGFR activation in pancreatic cancer cells.