Our previous study demonstrated that the herbal medicine, Oren-to, had antitumor effects on esophageal cancer cells (ECCs) in vitro. The purpose of this study was to examine which of the seven constituents of Oren-to had antitumor effects on esophageal cancer cells. MTT assay showed that, of the seven constituents, only the aqueous extract of Coptidis Rhizoma had potent inhibitory effect on the proliferation of two types of ECC lines, YES-3 and YES-4. In addition, the proliferation of all six types of ECC lines (YES-1 to YES-6) was inhibited in a dose-dependent manner (P<0.001 for all), when co-cultured at each concentration of Coptidis Rhizoma for 72 h. The ID50 of Coptidis Rhizoma for YES-1 to YES-6 was 2.2 microg/ml, 3.0 microg/ml, 0.25 microg/ml, 2.8 microg/ml, 2.5 microg/ml, and 0.5 microg/ml, respectively, berberine, one of protoberberine components of Coptidis Rhizoma, showed potent antitumor effects on all six types of ECC lines as well as Coptidis Rhizoma. In addition, the ID50 of berberine showed a positive correlation with that of Coptidis Rhizoma in six types of ECC lines examined (r2 = 0.763, P = 0.023). Cell cycle analysis of Coptidis Rhizoma-treated cancer cells showed the accumulation of cells in the G0/G1 phase and relative decrease of the S phase. These results support the possibility that the use of Coptidis Rhizoma containing abundant berberine may be useful as one of alternative therapies for esophageal cancers.