The development and progression of human cancer are believed to be due to the alterations of multiple genes or/and their protein products. For identifying the proteins associated with esophageal cancer, we analysed the protein profiles of 24 pairs of esophageal squamous cell carcinomas/matched adjacent normal epithelia. Microdissection of routinely unstained frozen sections was performed to purify cancerous and epithelial cells. The protein expression profiles were obtained by two-dimensional electrophoresis. Selected proteins dysregulated in tumors were identified by MALDI-TOF-MS. Three isoforms of annexin I were detected in normal esophageal mucosa and down-regulated in esophageal squamous cell carcinomas. RT-PCR analysis showed annexin I mRNA levels were significantly reduced in 17 out of 24 carcinomas. Immunohistochemistry demonstrated that annexin I appeared strong positive in all normal epithelia layers except basal cells. In cancer tissues, decreased expression of annexin I was observed in 12 out of 16 well differentiated tumors, 16 out of 17 moderately differentiated tumors, and 3 out of 3 poorly differentiated tumors as compared with the corresponding normal esophageal epithelia. There was a significant correlation between annexin I expression and the status of tumor differentiation. Well differentiated tumors presented stronger immunohistochemical reaction than moderately and poorly differentiated tumors. These data suggested that there existed three different isoforms of annexin I in normal esophageal epithelia, which may be the results of post-translational modification. Down-expression of three annexin I isoforms was a frequent event in esophageal carcinogenesis.