Multidrug resistance (MDR) is one of the main causes leading to the failure in cancer treatment.  Differential proteins between esophageal squamous cell carcinoma (ESCC) cell line EC9706 and its cis-diamminedichloroplatinum (CDDP)-resistant subline EC9706/CDDP revealed by quantitative analysis may provide deeper insights into the molecular mechanisms of MDR implicated in ESCC.  EC9706/CDDP was generated by exposure of its parental sensitive EC9706 to a step-wise increase of CDDP concentration during EC9706 cultivation.  The stable isotope labeling with amino acids in cell culture (SILAC) was used to label EC9706 and EC9706/CDDP with heavy and light medium, separately.  Mixed peptides derived from EC9706 and EC9706/CDDP were analyzed by high performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS/MS) and subsequently subjected to bioinformatics analysis to identify differential proteins between EC9706 and EC9706/CDDP.  Compared to parental EC9706, EC9706/CDDP manifested phenotypes of slow proliferation, cell pleomorphology, atypia and increased resistant-index 3.23.  Seventy-four differential proteins identified in the present study belongs to various families with multiple functions, such as cytoskeleton (20%), energy metabolism (11%), transcription regulation and DNA repair (11%), redox homeostasis (9.5%), protein biosynthesis and mRNA processing (12%), ribosome constituent (8.1%), molecular chaperone (8.1%), immunity/inflammation (5.4%), intracellular transport (5.4%) and nucleosome assembly (2.7%), which indicated that development of MDR is a complicated process involving dysregulation of multiple molecules and pathways.  The data is of great value for in-depth elucidation of molecular mechanisms of the MDR implicated in ESCC and may represent potential molecular targets for future therapeutic development.