The small guanosine triphosphatase (GTPase) Rho and its downstream effector Rho-associated kinase (ROCK) is one of a key mediator involved in controlling focal adhesions and the dynamics of actin stress fibers. The molecular mechanisms for the function of Rho/ROCK pathway leading to the progression in scirrhous gastric carcinoma cells have not been defined. The activation of RhoA in several gastric carcinoma cells was examined. The role of RhoA/ROCK pathway in the metastatic processes of gastric carcinoma cells, using a human scirrhous gastric cancer cell line, OCUM-2MD3 was investigated by in vitro adhesion and invasion assay. The effect of ROCK inhibitor, Y-27632 on the mRNA expression of the integrin family and MMP in gastric carcinoma cells was subsequently examined by Reverse transcriptional (RT)-PCR analysis. Finally, Random OCUM-2MD3 cell motility was evaluated using Time-lapse microscopy. ROCK inhibitor significantly increased the adhesion of OCUM-2MD3 cells to the extracellular matrix (ECM) protein matrigel. Further examination using ECM components showed enhanced binding ability was obtained only in laminin and Integrin subunits α3-integrin was clearly up-regulated by treatment with Y-27632 in OCUM-2MD3 cells. ROCK inhibitor also enhanced the invasion of OCUM-2MD3 cells through matrigel and the expression of membrane-type 1 matrix metalloproteinase (MT1-MMP). Time-lapse microscopy showed conversion of OCUM-2MD3 cells from round to more elongated morphology in the presence of Y-27632, suggesting that inhibition of RhoA/ROCK pathway undergo a so-called 'amoeboid to mesenchymal' transition. The fact that Rac1 inhibitor decreased the facilitated invasion by ROCK inhibitor suggested the possibility that increased invasion ability of OCUM-2MD3 cells was related to Rac activity. These data may suggest that RhoA/ROCK regulate plasticity of metastatic gastric carcinoma via mesenchymal-amoeboid transition, leading to provide new insights for designing a new and effective treatment for this type of refractory carcinoma.