In this study, we attempt to gain insights into the molecular mechanism underlying MDM2-mediated TGF-β resistance. MDM2 renders cells refractory to TGF-β by overcoming a TGF-β–induced G1 cell cycle arrest. Because the TGF-β resistant phenotype is reversible upon removal of MDM2, MDM2 likely confers TGF-β resistance by directly targeting the cellular machinery involved in the growth inhibition by TGF-β. Investigation of the structure-function relationship of MDM2 reveals three elements essential for MDM2 to confer TGF-β resistance in both mink lung epithelial cells and human mammary epithelial cells. One of these elements is the C-terminal half of the p53-binding domain, which at least partially retained p53-binding and inhibitory activity. Second, the ability of MDM2 to mediate TGF-β resistance is disrupted by mutation of the nuclear localization signal, but is restored upon coexpression of MDMX. Finally, mutations of the zinc coordination residues of the RING finger domain abrogates TGF-β resistance, but not the ability of MDM2 to inhibit p53 activity or to bind MDMX. These data suggest that RING finger-mediated p53 inhibition and MDMX interaction are not sufficient to cause TGF-β resistance and imply a crucial role of the E3 ubiquitin ligase activity of this domain in MDM2-mediated TGF-β resistance.