The Y-Box-Binding Protein-1 (YB-1) is known to regulate the processes of transcription, translation, cellular response to drug treatment and viral infection as well as DNA repair among others. As gastric cancer is a common cancer with a high incidence in countries in Asia, we evaluated the association of YB-1 with the malignant potential of gastric cancer cells in vitro. YB-1 mRNA expression levels were first determined by real-time RT-PCR in two adherent gastric cancer cell lines (viz., MKN7 and NUGC3 gastric cancer cells) and a normal GES-1 gastric epithelial cell line. Poorly differentiated NUGC3 gastric cancer cells were found to have the highest YB-1 gene expression among the adherent cells. YB-1 gene expression was also observed to be higher in non-adherent SNU5 gastric cancer cells compared to more aggressive SNU16 cells. Silencing of the YB-1 gene by siRNA in NUGC3 cells was associated with a significant reduction of the YB-1 protein by more than 55% as verified by Western blot analysis. Down-regulation of YB-1 protein expression was further demonstrated qualitatively by immunocytochemistry and immunofluorescence staining. Silencing of the YB-1 gene induced significant inhibition of cell migration in NUGC3 cells by 60% but did not influence cell invasion. Although epithelial-mesenchymal-transition (EMT) is known to be associated with the migratory phenotype in cancer cells, there was no change in the expression of EMT genes when YB-1 expression was modulated. YB-1 appears to have an integral role in cancer cell migration, a process which is important for gastric cancer metastasis. Anat Rec, 296:891–898, 2013. © 2013 Wiley Periodicals, Inc.