Mdm2 E3 ubiquitin ligase-mediated p53 degradation is generally accepted as the major mechanism for p53 regulation; nevertheless, the in vivo significance of this function has not been unequivocally established. Here, we have generated an Mdm2^Y487A knockin mouse; Mdm2^Y487A mutation inactivates Mdm2 E3 ligase function without affecting its ability to bind its homolog MdmX. Unexpectedly, Mdm2^Y487A/Y487A mice were viable and developed normally into adulthood. While disruption of Mdm2 E3 ligase function resulted in p53 accumulation, p53 transcriptional activity remained low; however, exposure to sublethal stress resulted in hyperactive p53 and p53-dependent mortality in Mdm2^Y487A/Y487A mice. These findings reveal a potentially dispensable nature for Mdm2 E3 ligase function in p53 regulation, providing insight that may affect how this pathway is targeted therapeutically.