To gain insights into the mechanisms of drug resistance in esophageal squamous cell carcinoma (ESCC), we employed proteomic techniques to study the global protein change of the multi-drug resistant ESCC cell line EC109/CDDP, which was established in our previous work, in comparison with its parental drug sensitive cell line EC109. By two-dimensional electrophoresis and mass spectrometry, we successfully identified 44 proteins with altered expression levels. These proteins are involved in endoplasmic reticulum stress response, metabolic process, DNA replication and repair, nucleotide binding, calcium binding, and cytoskeletal proteins. Among them, the differential expression levels of thioredoxin domain-containing protein 4 precursor and cystathionine gamma-lyase were further validated by Western blot and RT-PCR. Our present results lay foundation for future in-depth work on molecular mechanism of ESCC drug resistance, and aid in the identification and use of novel markers in clinical practice.