Objective: Direct interaction with cancer-associated fibroblasts triggers WNT5A expression in human gastric carcinoma (GC) cells. In this study, we performed gene transduction experiments to investigate the significance of WNT5A in the GC tumor microenvironment. Methods: Gene transduction (pWNT5A and shWNT5A) was performed in human GC-derived MKN-7 cells. Altered gene expression was examined by RT-PCR and cDNA microarray analysis. Immunohistochemical examination was carried out in human GC tissues. Results: Transduction of exogenous WNT5A expression into MKN-7 cells upregulated genes related to the epithelial-mesenchymal transition (EMT) and cancer stem cells (CSCs), and the pWNT5A transfectant showed high tumorigenicity in vivo. These results were confirmed by knockdown experiments using a lentivirus expressing shWNT5A. A cDNA microarray analysis suggested that depletion of endogenous WNT5A downregulated genes involved in intracellular signaling, chemokine-cytokine interaction and focal adhesion. High levels of WNT5A expression were observed in 66% of GC cases, with significant correlation with histological type. Interestingly, in intestinal-type GCs, WNT5A expression was detected in the periphery of tumor nests. Conclusions: WNT5A regulates the induction of EMT and the maintenance of CSC properties in MKN-7 cells. WNT5A may play an important role in constructing an advantageous tumor microenvironment for the progression and development of human GC.