Conditionally replicative adenoviruses (CRAd) are under investigation as anticancer agents. Previously, we found that the CRAd AdDelta24-p53, expressing the p53 tumor suppressor protein from its genome, more effectively killed most human cancer cells than did its parent AdDelta24. However, a minority of cancer cell lines poorly responded to the oncolysis-enhancing effect of p53. Here we show that refractory cell lines expressed high levels of the major negative p53 regulator murine double minute 2 (MDM2). To obviate MDM2-mediated inactivation of CRAd-encoded p53, we constructed the new CRAd AdDelta24-p53(14/19) encoding a p53 variant incapable of binding to MDM2. AdDelta24-p53(14/19) was approximately 10 times more effective than AdDelta24-p53 in killing cancer cell lines with high levels of human MDM2, but not cells with low MDM2. This finding supports the notion that exogenous expression of functional p53 augments the anticancer efficacy of CRAds. In addition, it confirms that high MDM2 expression is a molecular determinant of resistance against oncolysis enhancement by exogenous wild-type p53. Moreover, it shows that efficacy enhancement by restoration of functional p53 can also be accomplished in cancer cells expressing a p53 inhibitor. This further expands the utility of CRAds expressing functional p53 variants for effective virotherapy of cancer and thus their possible contribution to the advancement of individualized molecular medicine.