Rho-associated protein kinase (ROCK), a molecular switch, modulates cellular functions in many cancers, such as hepatocellular, breast, colon cancers, etc. However, little is known the effect of ROCK on cell adhesion and mobility in esophageal squamous cell cancer (ESCC), one of the most diagnosed cancers in China. In this study, Y-27632 was used to specifically block ROCK activity in ESCC cells. Adhesion of ESCC cells was detected by homotypic and heterotypic adhesion assay together with examination of E-cadherin expression. Motility of ESCC cells changes were examined by detection of phosphorylated cofilin and observed under confocal microscopy, respectively. We found that Y-27632 increased both heterotypic and homotypic adhesion, and the expression of E-cadherin; decreased phosphorylated cofilin resulting in actin rearrangement in ESCC cells. All these findings indicate that ROCK signaling pathway plays an important role in cell adhesion and mobility, suggesting that it may be used as a potential target for therapy of ESCC.