Osteosarcoma (OS) in human patients is characterized by genetic alteration of TP53. Osteoprogenitor-specific p53-deleted mice (OS mice) have been widely used to study the process of osteosarcomagenesis. However, the molecular mechanisms responsible for the development of OS upon p53 inactivation remain largely unknown. In this study, we detected prominent RUNX3/Runx3 expression in human and mouse p53-deficient OS. Myc was aberrantly upregulated by Runx3 via mR1, a consensus Runx site in the Myc promoter, in a manner dependent on p53 deficiency. Reduction of the Myc level by disruption of mR1 or Runx3 knockdown decreased the tumorigenicity of p53-deficient OS cells and effectively suppressed OS development in OS mice. Furthermore, Runx inhibitors exerted therapeutic effects on OS mice. Together, these results show that p53 deficiency promotes osteosarcomagenesis in human and mouse by allowing Runx3 to induce oncogenic Myc expression.