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p53 is a tumor suppressor that responds to a variety of stresses such as oncogenes and DNA damage by activating its transcriptional targets to allow repair or elimination of damaged cells. In the absence of stress signals, p53 needs to be kept in check and this is achieved by the E3 ligase MDM2. For tumors that retain wild-type p53, therapeutic strategies aimed at removing the inhibitory activity of MDM2 on p53 are under development and to date have focused on drugs that prevent the binding of p53 to MDM2. Here, we report the analysis of a group of synthetic analogs derived from 5-deazaflavin compounds previously identified in a screen as inhibitors of MDM2 autoubiquitination. Using measurement of surface plasmon resonance, we demonstrated that active 5-deazaflavin analogs bind to the MDM2 RING, whereas inactive compounds show no binding. In cellular assays, these active MDM2 RING binding compounds inhibited the ubiquitination of p53, stabilized p53, led to increased expression of p53 targets and caused corresponding cell cycle effects. Deazaflavin analogs therefore function to activate p53 through a novel mechanism, by inhibiting the E3 ligase activity of MDM2 in a manner that involves binding to the MDM2 RING.