Members of the forkhead class O (FOXO) transcription factors are tumor suppressors and key molecules that control aging and lifespan. The stability of mammalian FOXO proteins is controlled by proteasome-mediated degradation but general ubiquitin E3 ligases for FOXO factors remain to be defined. The current studies demonstrate that MDM2 bound to FOXO1 and FOXO3A and promoted their ubiquitination and subsequent degradation, a process apparently dependent on FOXO phopshorylation at PKB sites and on the E3 ligase activity of MDM2. The binding occurred between endogenous proteins and was involved the forkhead box of FOXO1 and the region of MDM2 that controls its cellular localization. MDM2 promoted the ubiquitination of FOXO1 in vitro in a cell free system. Knocking down MDM2 by siRNA caused the accumulation of endogenous FOXO3A protein, and enhanced the expression of FOXO target genes. In addition, MDM2 promoted the transcriptional activity of FOXO in a transient transfection system. In cells stably expressing a temperature sensitive mutant p53, activation of p53, by shifting to permissive temperatures led to MDM2 induction and the degradation of endogenous FOXO3A. These data suggested that MDM2 acts downstream of p53 as an E3 ubiquitin ligase to promote the degradation of mammalian FOXO factors.