MDM2 belongs to a class of ring-finger domain-containing ubiquitin ligases that mediate the proteasomal degradation of numerous proteins, including themselves. Arguably, the most important substrate of MDM2 is p53, which controls cell cycle progression and apoptosis. MDM2 and p53 are parts of a feedback regulatory loop whose perturbations are often present in cancer and are targets for anticancer drug development. We found that the natural product, small-molecule anti-inflammatory agent parthenolide (PN), which is actively being investigated as a potential therapeutic for many human cancers, induces ubiquitination of MDM2 in treated cells, resulting in the activation of p53 and other MDM2-regulated tumor-suppressor proteins. Using cells with functional gene deletions and small interfering RNA knockdown studies, we found that these effects required the DNA damage transducer ataxia telangiectasia mutated. The effects of PN on tumor suppressor activation were comparable with that of nutlin-3a, a recently developed small molecule that was designed to interfere with the interaction between MDM2 and p53 but does not promote MDM2 ubiquitination. Our study illustrates an alternative approach for controlling MDM2 and p53 activities and identifies an additional critically important cancer pathway affected by PN.