Tight junctions are commonly disrupted in cancer cells, including gastric cancer. Various growth factors have been reported to affect the localization of tight junction-associated proteins such as ZO-1 and occludin. We investigated the effect of epidermal growth factor (EGF), a growth factor that is often overexpressed in gastric cancer, and fetal bovine serum (FBS) on the localization of ZO-1 and occludin in a gastric cancer cell line. In the poorly differentiated gastric cancer cell line TMK-1, immunohistochemistry demonstrated that ZO-1 and occludin were predominantly localized to the cytoplasm, although there was some weak expression at the cell-cell contact. When the medium was replaced with fresh medium containing 10% FBS, ZO-1 and occludin were rapidly translocated from the cytosol to the cell-cell contact. A similar effect was seen in EGF exposure. These effects induced by FBS or EGF were attenuated in the presence of protein kinase C (PKC) inhibitors calphostin C and bisindolylmaleimide I, but not another PKC inhibitor Gö6976, PD98059 (MAPK inhibitor), LY294002 (PI3 kinase inhibitor) or KT5720 (protein kinase A inhibitor). These results suggest that serum-derived factors, including EGF, can rapidly alter the localization of ZO-1 and occludin via a protein kinase C signaling pathway in TMK-1 gastric cancer cells.