Overexpression of mutant p53 is a common theme in human tumors, suggesting a tumor-promoting gain-of-function for mutant p53. To elucidate whether and how mutant p53 acquires its gain-of-function, mutant p53 is inducibly knocked down in the SW480 colon cancer cell line, which contains mutant p53(R273H/P309S), and the MIA PaCa-2 pancreatic cancer cell line, which contains mutant p53(R248W). We found that knockdown of mutant p53 markedly inhibits cell proliferation. In addition, knockdown of mutant p53 sensitizes tumor cells to growth suppression by various chemotherapeutic drugs. To determine whether a gene involved in cell growth and survival is regulated by mutant p53, gene expression profiling analysis was performed and showed that the expression level of Id2, a member of the inhibitor of differentiation (Id) family, was markedly increased upon knockdown of mutant p53. To confirm this, Northern blot analysis was performed and showed that the expression level of 1d2 was regulated by various mutant p53s in multiple cell lines. In addition, we found that the Id2 promoter is responsive to mutant but not wild-type p53, and mutant p53 binds to the Id2 promoter. Consistent with these observations, expression of endogenous Id2 was found to be inhibited by exogenous mutant p53 in p53-null HCT116 cells. Finally, we showed that knockdown of Id2 can restore the proliferative potential of tumor cells inhibited by withdrawal of mutant p53. Together, these findings suggest that one mechanism by which mutant p53 acquires its gain-of-function is through the inhibition of Id2 expression. © 2008 American Association for cancerResearch.