Numerous studies indicate that p300 acts as a key transcriptional cofactor in vivo, at least, in part, by modulating activities of p53 by acetylation. Nevertheless, the regulation of the p53-p300 interplay is not completely understood. Here, we have identified the DEAD (Asp-Glu-Ala-Asp) box RNA helicase 24 (DDX24) as a novel regulator of the p300-p53 axis. We found that DDX24 interacts with p300, and this interaction leads to suppression of p300-mediated acetylation of p53. Notably, RNA interference-mediated knockdown of endogenous DDX24 significantly increases the acetylation levels of endogenous p53 in human cancer cells and subsequently promotes p53-mediated activation of its transcriptional targets such as p21 and p53 upregulated modulator of apoptosis (PUMA). In contrast, DDX24 expression inhibits the p300-p53 interaction and suppresses p300-mediated acetylation of p53. Moreover, DDX24 is overexpressed in human cancer cells and reduction of DDX24 protein levels by RNA interference induces cell cycle arrest and senescence in a p53-dependent manner. These results reveal DDX24 as an important regulator of p300 and suggest that the modulation of the p53-p300 interplay by DDX24 is critical in controlling p53 activities in human cancer cells.