Esophageal adenocarcinoma, currently the seventh leading cause of cancer-related death, has been associated with the presence of Barrett's metaplasia. The malignant potential of Barrett’s metaplasia is evidenced by ultimate progression of this condition to invasive adenocarcinoma. We have utilized liquid phase separation of proteins with chromatofocusing in the first dimension and nonporous reverse phase (NPS-RP) HPLC in the second dimension, followed by ESI-TOF mass spectrometry to identify proteins differentially expressed in Barrett’s metaplasia as compared to esophageal adenocarcinoma isolated from the same patient. Approximately 300 protein bands were detected by mass mappings and 40 differentially expressed proteins were identified by µLC-MS/MS. Among them, Rho GDP-dissociation inhibitor 2, alpha-enolase, lamin A/C and nucleoside diphosphate kinase A were demonstrated to be up-regulated in both mRNA and protein expression in esophageal adenocarcinomas relative to Barrett’s metaplasia. Candidate proteins were examined at the mRNA level using high density oligonucleotide microarrays. The cellular expression patterns were verified in both esophageal adenocarcinomas and in Barrett’s metaplasia by immunohistochemistry (IHC). Additionally, several other interesting differentially expressed proteins were identified, including calgranulin B and tropomyosin 1. These differentially expressed proteins may have utility as useful biomarkers of disease progression in esophageal adenocarcinoma.