Esophageal cancer (EC) persists to be a leading cancer-related death in northern China. Clinical outcome of EC is the most dismal among many types of digestive tumors because EC at early stage is asymptomatic. The current study used 2-DE-based proteomics to identify differentially expressed proteins between esophageal cancer cell lines and immortal cell line. Fifteen proteins were identified with differences of more than five folds, comprising the down-regulation of annexin A2, histone deacetylase 10 isoform beta and protein disulfide-isomerase ER-60 precursor, and the up-regulation of heat shock 70 kDa protein 9B precursor, solute carrier family 44 Member 3, heterogeneous nuclear ribonucleoprotein L (hnRNP L), eukaryotic translation initiation factor 4A isoform 2, triosephosphate isomerase1 (TPI), peroxiredoxin1 (PRX1), forminotransferase cyclodeaminase form (FTCD), fibrinogen gamma-A chain precursor, kinesin-like DNA binding protein, lamin A/C, cyclophilin A (CypA), and transcription factor MTSG1. Expression pattern of annexin A2 was verified by Western blotting, immunocytochemistry and immunohistochemistry analysis. The implication of these protein alterations correlated to the esophageal malignant transformation is discussed.