RUNX3, a runt-related transcription factor, has a crucial role in dorsal root ganglion neurogenesis. Recent studies have suggested that RUNX3 acts as a tumor suppressor in stomach, colon and breast cancer. However, the biological role of RUNX3 in neuroblastoma remains elusive. Here we report that high levels of RUNX3 expression contribute to the favorable outcome in patients with neuroblastoma, whereas low levels of RUNX3 expression result in poor outcome. Array-based analysis suggested that the allelic loss at chromosome 1p36 is one of the reasons why expression of RUNX3 is downregulated in advanced neuroblastomas. Interestingly, the several patients survived from neuroblastoma with both high mRNA expressions of MYCN and RUNX3, suggesting that RUNX3 high expression might overcome the aggressive behavior of MYCN. Exogenous expression of RUNX3 strongly inhibits cell proliferation and migration in neuroblastoma cell lines. Furthermore, RUNX3 reduces the stability of MYCN protein in MYCN-amplified neuroblastoma cell lines, and this RUNX3-mediated MYCN degradation may depend on the physical interaction between RUNX3 and MYCN. Thus, our findings provide a tumor-suppressing mechanism by which RUNX3 inhibits the MYCN activity in neuroblastoma.