Gastric cancer (GC) is the one of the most common types of cancer in Asia. To better understand the molecular mechanisms underlying GC, and to seek new markers of tumor progression, we used a proteomics strategy to analyze the protein expression patterns in matched pairs of GC tissue and normal gastric mucosa of 8 GC patients. Comparative proteomic analysis, using two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), revealed that 32 protein spots showed a >2-fold difference in intensity between tumor and normal tissues. Twenty-six proteins were up-regulated and 6 proteins were down-regulated in tumor tissue compared to control. Western blot analysis confirmed differential expression for 9 proteins, including AGR2, ENO1, GDI2, GRP78, GRP94, PPIA, PRDX1, PTEN and VDAC1. Immunohistochemical staining of a tissue microarray, derived from 145 GC patients, with antibodies for each of the 9 proteins demonstrated a significant association between the level of protein immunostaining and the clinical features of the disease in the donor. The identified proteins were functionally classified using bioinformatics methods, showing that the 9 proteins identified were related to BCL2, BAX, ERBB2 and CASP3 proteins and involved in the process of apoptosis. These proteomic data provide potentially valuable insights into both the biology of GC and the identity of biomarkers for tumor progression. We propose ENO1, GRP78, GRP94, PPIA, PRDX1 and PTEN as potential GC biomarkers.