Fasudil, an inhibitor of Rho kinase, is known to suppress tumorigenicity and cancer metastasis. However, the underlying molecular mechanisms of how fasudil suppresses cell metastasis have not been fully elucidated. The purpose of this study was to determine the effects of fasudil on migration and cancer growth and to evaluate Rho kinase activity in the 95-D lung carcinoma cell line. The cytotoxic effect of drugs on 95-D cells was measured by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Treatment with fasudil inhibited the growth of 95-D cells in a dose-dependent manner, and the IC50 of fasudil was approximately 0.79mg/ml (95% confidence limits (CL): 0.58–1.11mg/ml). The total amounts of active MMP2 and MMP9 per microgram of protein when treated with 0.75mg/ml fasudil and using the gelatinase assay were decreased compared with the control group by about 22.7% (P<0.05) and 65.9% (P<0.01) respectively. Although ABCC1, ABCC3, ABCA3, and ABCC5 were over-expressed at the mRNA level, ABCE1 was the only transporter responsible for resistance in this study. We also found that myosin phosphatase targeting subunit 1 (MYPT1) phosphorylation at Thr-696, which provided direct evidence of Rho kinase activity, was reduced by 29.4% in response to fasudil compared with the control group (P<0.05). Taken together, our findings show that fasudil prevents cancer metastasis by inhibiting the Rho/Rho kinase pathway and that the ABCE1 gene was involved in the migration of 95-D cells.