Although it has been shown that the gastric tumor suppressor RUNX3 has a growth inhibitory activity, the precise molecular mechanisms behind RUNX3-mediated tumor suppression remained unclear. In this study, we found that RUNX3 is closely involved in DNA damage-dependent phosphorylation of tumor suppressor p53 at Ser-15 and acts as a co-activator for p53. The small interference RNA-mediated knockdown of RUNX3 inhibited adriamycin (ADR)-dependent apoptosis in p53-proficient cells but not in p53-deficient cells in association with a significant reduction of p53-target gene expression as well as phosphorylation of p53 at Ser-15. In response to ADR, RUNX3 was induced to accumulate in the cell nucleus and co-localized with p53. Immunoprecipitation experiments demonstrated that RUNX3 forms a complex with p53 in cells. In vitro pulldown assays revealed that the COOH-terminal portion of p53 is required for the interaction with RUNX3. Forced expression of RUNX3 enhanced p53-mediated transcriptional activation. Additionally, RUNX3 had an ability to induce the phosphorylation of p53 at Ser-15, thereby promoting p53-dependent apoptosis. Intriguingly, RUNX3 interacted with phosphorylated forms of ataxia telangiectasia-mutated in response to ADR; however, it did not affect the extent of DNA damage. From the clinical point of view, coordinated p53 mutation and decreased expression of RUNX3 in 105 human lung adenocarcinomas were significantly associated with the poor outcome of patients (p = 0.0203). Thus, our present results strongly suggest that RUNX3 acts as a novel co-activator for p53 through regulating its DNA damage-induced phosphorylation at Ser-15 and also provide a clue to understanding the molecular mechanisms underlying RUNX3-mediated tumor suppression.