In our previous investigations, mitogen-activated protein kinase kinase 2 (MEK2)/extracellular signal-regulated kinase 2 (ERK2) signaling pathway was found to be correlated with the cell dissociation induced by dissociation factor (DF) in pancreatic cancer cells. In this study, the expressions of epidermal growth factor receptor (EGFR), phosphorylated EGFR (p-EGFR), and its downstream kinases MEK1/2 and ERK1/2, were analyzed to clarify the regulatory mechanism of cell dissociation in pancreatic cancer cells. Two hamster (PC-1.0 and PC-1) and two human (AsPC-1 and Capan-2) pancreatic cancer cell lines were used. Immunocytochemical study was performed using anti-EGFR, p-EGFR, phosphorylated MEK1/2 (p-MEK1/2), and phosphorylated ERK1/2 (p-ERK1/2) antibodies. DF-treatment markedly induced the expressions of EGFR, p-EGFR, p-MEK1/2, p-ERK1/2, as well as the dissociation of cell colonies in PC-1 and Capan-2 cells. In contrast, AG1478 (an EGFR inhibitor) treatment significantly induced the cell aggregation in PC-1.0 and AsPC-1 cells which usually grew as single cells, but strongly suppressed the expressions of EGFR, p-EGFR, p-MEK1/2, and p-ERK1/2. These observations demonstrate that activation of EGFR is closely involved in cell dissociation in pancreatic cancer through activating MEK/ERK signaling pathway.