We isolated a metastasizing human esophageal squamous cell carcinoma (SCC) cell line, T.Tn-AT1, from a parental non-metastasizing cell line, T.Tn, by in vitro selection and by use of a nude mouse orthotopic inoculation model. Then, we compared the expression profiles of 9206 genes in T.Tn-AT1 and T.Tn by cDNA microarray analysis. The gene expression profiles of T.Tn and T.Tn-AT1 were very similar, and only 34 genes showed more than 3-fold differential expression. Among the 34 genes, 29 genes were down-regulated and only 5 genes were up-regulated in T.Tn-AT1 cells. Subsequently, we confirmed the expression levels of 14 of the 34 genes in T.Tn and T.Tn-AT1 cells by means of reverse transcription-polymerase chain reaction. The expression of 8 genes (KAL1, HPGD, NDN, REG1A, CXCR4, SPOCK, DIAPH2 and AIF1) was down-regulated and that of one gene (VNN2) was up-regulated in T.Tn-AT1 cells. These 9 genes encoded proteins associated with metastatic processes, such as adhesion, migration, inflammation, proliferation, and differentiation. Thus, these genes might regulate the metastasis of esophageal SCC, and could be predictive markers for lymph node metastasis of esophageal SCC.