The vast majority of p53 missense mutants lose the wild-type (wt) function and/or exert ‘dominant-negative’ effects on their wt counterpart. Here, we identify a novel form of p53 mutation with an extended C-terminus (p53 long C-terminus, p53LC) in a variety of human cancers. Interestingly, the two representative mutants (named ‘p53-374*48’ and ‘p53-393*78’) as tested in this study show both loss-of-function and dominant-negative phenotypes in cell proliferation and colony formation assays. Mechanistically, p53LCs interact with and retain wt p53 in the cytoplasm and prevent it from binding to the promoters of target genes, consequently inhibiting its transcriptional activity. Also, p53LCs are very stable, though not acetylated in cells. Remarkably, the p53LCs can desensitize wt p53-containing cancer cells to p53-activating agents. Together, our results unveil a longer form of p53 mutant that possesses a dominant-negative effect on its wt counterpart, besides losing its wt activity.