The aim of this study was to establish a stable, drug-resistant esophageal squamous cell carcinoma (ESCC) cell line. The human ESCC cell line EC109 was exposed to cisplatin (CDDP) by pulse treatment to select for the drug-resistant subline, EC109/CDDP cells. The MTT assay was used to test the drug resistance of EC109 and EC109/CDDP cells. In addition, cellular morphological changes were observed using microscopy and the growth curves of the two cell lines were drawn to calculate the doubling time. Furthermore, cell cycle distribution was analyzed by flow cytometry. Finally, RT-PCR was performed to determine the mRNA expression levels of drug-resistant-related genes, multidrug resistance 1 (MDR1), multidrug resistance-associated protein 1 (MRP1), ATP-binding cassette, sub-family G, member 2 (ABCG2), lung resistance protein (LRP), and glutathione S-transferase (GST)-pi in both cell lines. EC109/CDDP cells exhibited increased resistance to CDDP, carboplatin, 5-fluorouracil, taxol, navelbine, irinotecan and etoposide, and changes in morphology, doubling time, and cell cycle distribution were detected as compared with EC109 cells. Although there was no significant difference in MRP1, ABCG2, LRP, and GST-pi expression, MDR1 expression in EC109/CDDP cells was lower than that of EC109 cells. EC109/CDDP cells are a stable, multidrug-resistant ESCC cell line and could serve as an important tool for further research concerning ESCC drug resistance.