Gastric cancer is one of the leading causes of cancer mortality and its malignancy, resulting from disseminated cancer cells of diffuse type, is clinically manifested as metastases to the liver and peritoneum. The aim of the present study was to identify putative tumor metastasis-associated genes in human gastric cancer cells of diffuse type. An MKN45 cell line constitutively expressing green fluorescent protein (MKN45-GFP) was established and selected using the Transwell® system for invasive sublines MKN45-GFP-4, MKN45-GFP-10 and MKN45-GFP-12. MKN45-GFP-10 and MKN45-GFP-12 are highly invasive compared to the others. The mRNA levels were measured with cDNA microarrays and correlated with their invasion abilities in these sublines. Many of the genes identified with a positive or negative correlation are associated with angiogenesis, cell cycle, cytoskeleton and cell motility, protease and cell adhesion, as well as cellular signal transduction. In particular, novel genes without known functions were also noted. RT-PCR and western blot analyses were applied to verify the expression of selective genes. Following orthotopical intraperitoneal implantation, MKN45-GFP-12 demonstrated significantly higher in vivo tumor malignancies than parental MKN45-GFP in ascites induction and liver -invasion in mice. We have identified putative gastric tumor metastasis-associated, as well as novel genes. These genes and their protein products are to be further explored for their functional roles associated with tumor metastasis. The molecular profiles of these identified genes, gene transcripts and proteins in the patient specimens are likely to be useful biomarkers for diagnostic, therapeutic and/or prognostics. Most importantly, they may be used as molecular targets for the discovery of antitumor drugs against human gastric cancer metastasis.