Abstract Gα12/13, which belongs to the Gα12 family, participates in the regulation of diverse physiologic processes. In view of the control of Gα12/13 in cell proliferation, this study investigated the role of Gα12/13 in the regulation of p53 and mdm4. Immunoblotting and immunocytochemistry revealed that p53 was expressed in control embryonic fibroblasts and was largely localized in the nuclei. Gα12 deficiency decreased p53 levels and its DNA binding activity, accompanying p21 repression with Bcl2 induction, whereas Gα13 deficiency exerted weak effects. Gα12 or Gα13 deficiency did not change p53 mRNA expression. ERK1/2 or Akt was not responsible for p53 repression due to Gα12 deficiency. Mdm4, a p53-stabilizing protein, was repressed by Gα12 deficiency and to a lesser extent by Gα13 deficiency, whereas mdm2, PTEN, β-catenin, ATM, and Chk2 were unaffected. p53 accumulation by proteasomal inhibition during Gα12 deficiency suggested the role of Gα12 in p53 stabilization. Constitutively active Gα12 (Gα12QL) or Gα13 (Gα13QL) promoted p53 accumulation with mdm4 induction in MCF10A cells. p53 accumulation by mdm4 overexpression, but no mdm4 induction by p53 overexpression, and small interfering RNA knockdown verified the regulatory role of mdm4 for p53 downstream of Gα12/13. In control or Gα12/Gα13-deficient cells, genotoxic stress led to p53 accumulation. At concentrations increasing the flow cytometric pre-G1 phase, doxorubicin or etoposide treatment caused serine phosphorylations in Gα12−/− or Gα12/13−/− cells, but did not induce mdm4. Gα12/13QL transfection failed to phosphorylate p53 at serines. Our results indicate that Gα12/13 regulate basal p53 levels via mdm4, which constitutes a cell signaling pathway distinct from p53 phosphorylations elicited by genotoxic stress. (Mol Cancer Res 2007;5(5):473–84)