Gastric cancer is one of the most frequently diagnosed malignancies in the world. The gene expression profile and molecular grouping of gastric cancer has been a challenging task due to its inherent complexity and variation among individuals.To determine the molecular mechanism associated with gastric carcinogenesis.We analyzed the gene expression profiles of 20 cancerous tissues and their tumor-adjacent tissue from patients with gastric cancer by using a 14 K cDNA microarray. The differentially expressed genes and their products were verified by semiquantitative reverse transcription PCR (RT-PCR), western blotting and immunohistochemistry of gastric cancer and normal tissue samples.A total of 69 genes were found to be differentially regulated in the cancerous tissue. Among them, genes such as CDH17, ETV4, S100A6, S100A11, Ephb4, and KLK10 were confirmed by RT-PCR to be up-regulated, while genes such as NK4 and PPP2R1B were down-regulated. Western blotting and immunohistochemistry indicated that Ephb4 was over-expressed and localized to the cytoplasm of gastric cancer cells. Moreover, Ephb4 protein was observed as being significantly related to tumor size and pN category (p = 0.001 and 0.007, respectively).These newly identified genes might provide a valuable resource for understanding the molecular mechanism associated with the carcinogenesis of gastric cancer and for finding potential diagnostic markers of gastric cancer.