Loss of RUNX3 expression is frequently observed in gastric cancer and is highly associated with lymph node metastasis and poor prognosis. However, the underlying molecular mechanisms of gastric cancer remain unknown. In this study, we found that the protein levels of RUNX3 and osteopontin (OPN) are inversely correlated in gastric cancer clinical specimens and cell lines. Furthermore, similar inverse trends between RUNX3 and OPN messenger RNA (mRNA) expression were demonstrated in six out of seven normal-tumor-paired gastric cancer clinical specimens. In addition, low RUNX3 and high OPN expression were associated with poor prognosis in gastric cancer patients. Ectopic expression of green fluorescent protein-RUNX3 reduced OPN protein and mRNA expression in the AGS and SCM-1 gastric cancer cell lines. In contrast, knockdown of RUNX3 in GES-1, a normal gastric epithelial cell line, increased OPN expression. Although three RUNX3-binding sequences have been identified in the OPN promoter region, direct binding of RUNX3 to the specific binding site, -142 to -137bp, was demonstrated by chromatin immunoprecipitation assay. The binding of RUNX3 to the OPN promoter significantly decreased OPN promoter activity. The knockdown of OPN or overexpression of RUNX3 inhibited cell migration in AGS and SCM-1 cells; however, the coexpression of RUNX3 and OPN reversed the RUNX3-reduced migration ability in AGS and SCM-1 cells. In contrast, the knockdown of both RUNX3 and OPN inhibited RUNX3-knockdown-induced migration of GES-1 cells. Together, our data demonstrated that RUNX3 is a transcriptional repressor of OPN and that loss of RUNX3 upregulates OPN, which promotes migration in gastric cancer cells.