p38 mitogen-activated protein kinases (MAPK) contribute to the loss of cell-cell contact and the round cell shape characteristic of poorly differentiated gastric cancer. In the present study it is demonstrated that phospho-p38 MAPK level significantly increased in poorly differentiated gastric cancers in comparison to differentiated cancers and normal gastric mucosa by immunohistochemistry. Next, the pathophysiological roles of p38 MAPK activation were investigated in differentiated gastric cancer cell lines MKN7 and MKN28 and poorly differentiated gastric cancer cell lines KATO-III and MKN45 cells by incubating with specific p38 inhibitor SB203580 or inactivating analog SB202474. The distribution of F-actin on phalloidin staining was identified as fine cytoskeletal filaments in MKN7 and MKN28, but as dense membranous accumulation in KATO-III and MKN45 cells. The treatment with SB203580 but not SB202474 reduced irregular accumulation of F-actin in KATO-III and MKN45 cells. The expression of E-cadherin, ZO-1, occludin and claudin 4 was higher in MKN7 and MKN28 than KATO-III and MKN45 cells. The expression of E-cadherin in KATO-III cells was increased following treatment with SB203580, suggesting the suppression of E-cadherin at the transcriptional level independent of its genetic alterations. Thus, p38 MAPK signaling might contribute to the acquisition of malignant properties in poorly differentiated phenotypes.