Objective To investigate the effect of Rho kinase (ROCK) on the morphology and migration of cultured Schwann cells. Methods Primary culture Schwann cells were performed and the purity of Schwann cell was identified by immunofluorescence. Single-cell migration assay for Schwann cells was established. Based on this assay, a gradient of ROCK inhibitor Y27632 was produced in front of migrating Schwann cells. The behavior of morphology and motility in Schwann cells by Y-27632 were recorded based on time-lapse imaging. Results Immunofluorescence showed that cultured cells were of high purity of Schwann cell and single-cell migration assay was suitable for studying the morphology and motility of Schwann cells. Y-27632 dramatically promoted the more braches, thinner and longer processes, and inhibited Schwann cell migration. Conclusion ROCK can maintain the morphology and regulate the motility of Schwann cells.