Vorinostat (suberoylanilide hydroxamic acid, SAHA) represents a new class of highly potent histone deacetylase (HDAC) inhibitors that cause growth arrest, differentiation, and apoptosis of many tumor types in vitro and in vivo. RUNX3, a gastric tumor suppressor, is epigenetically silenced in gastric cancer cells. This study investigates the role of RUNX3 in vorinostat-induced suppression of gastric cancer cell growth. RUNX3 was up-regulated by vorinostat in gastric cancer cell lines not expressing RUNX3. In terms of cell viability, the mean IC50 of vorinostat in RUNX3-negative cells was significantly lower than that seen in RUNX3-positive cells, indicating that the former are more sensitive to vorinostat in terms of growth arrest than are RUNX3-positive lines. The mechanism underlying this difference was found to be reactivation of RUNX3 expression by vorinostat and concomitant increase in acetylated histone H3 in the promoter region of RUNX3. Using three RUNX3-negative cell lines, we determined the contribution of RUNX3 reactivation to growth inhibition and induction of apoptosis following treatment of cells with vorinostat and found that upregulated RUNX3 was significantly responsible for tumor suppressive activities.