Background/Aims: Different differentiations of-cancer have resulted in its unique biological characteristics. We screen and appraise differentially expressed proteins in different differentiated gastric adenocarcinoma with comparative proteomics technology in order to find regulatory factors of tumor differentiation related and finally reach the purpose of tumor differentiation reversal. Methodology: With two-dimensional fluorescence difference gel electrophoresis (2-D DIGE) and liquid chromatography in conjunction with tandem mass spectrometry (LC-MS/MS), the differentially expressed proteins from 8 patients with different differentiated gastric adenocarcinoma were identified and some factors identified were verified with application of QPCR and Western blot techniques. Results: Significant differences in 35 protein spots were found and 48 kinds of proteins were identified. Other than structural proteins and non-specific protein, six possible proteins associated with tumor differentiation were determined - the serine protease inhibitor B1 (serine protease inhibitor, clade B, member 1, SERPINB1), calcium-phospholipid binding protein III (annexin A3), transcription factor Nm23-H1, adenine phosphoribosyl-transferase enzyme APRT (Adenine Phosphoribosyltransferase in APO and AMP), glutathione S-transferase P1-1 (GST-pi-1), antimicrobial peptides Dermcidin-IL. The identified SERPINB1, annexin A3, Nm23-H1 and APRT were verified, confirming the expression of these factors was in line with proteomics identification. Conclusions: Protein expression in different differentiated gastric adenocarcinoma was varied.