Abstract Strategies to treat cancer by restoring p53 tumor suppressor functions are being actively investigated. These approaches range from expressing an exogenous p53 gene in p53 mutant cancers to antagonizing a p53 inhibitor in p53 wild-type (WT) cancer cells. In addition, exogenous p53 is used to strengthen the anticancer efficacy of oncolytic adenoviruses. Many cancers express high levels of the major negative regulator of p53, mouse double minute 2 (MDM2) protein. Recently, a novel class of highly potent and specific MDM2 antagonists, the Nutlins, was identified. We envisioned that Nutlins could protect both endogenous and exogenous p53 from MDM2-mediated inactivation. We therefore investigated treating human cancer cells with a combination of adenovirus-mediated p53 gene therapy and Nutlin. Combination treatment resulted in broadly effective cell kill of p53 WT and p53-negative cancer cells. Cytotoxicity was associated with profound cell cycle checkpoint activation and apoptosis induction. We also tested Nutlin in combination with oncolytic adenoviruses. Nutlin treatment accelerated viral progeny burst from oncolytic adenovirus-infected cancer cells and caused an estimated 10- to 1,000-fold augmented eradication of p53 WT cancer cells. These findings suggest that Nutlins are promising compounds to be combined with p53 gene therapy and oncolytic virotherapy for cancer. [Mol Cancer Ther 2007;6(5):1552–61]