The F box protein Skp2 is oncogenic, and its frequent amplification and overexpression correlate with the grade of malignancy of certain tumors. Conversely, depletion of Skp2 decreases cell growth and increases apoptosis. Here, we show that Skp2 counteracts the transactivation function of p53 and suppresses apoptosis mediated by DNA damage or p53 stabilization. We demonstrate that Skp2 forms a complex with p300 through the CH1 and the CH3 domains of p300 to which p53 is thought to bind and antagonizes the interaction between p300 and p53 in cells and in vitro. As Skp2 antagonizes the interaction between p300 and p53, Skp2 suppresses p300-mediated acetylation of p53 and the transactivation ability of p53. Conversely, ectopic expression of p300 rescues the transactivation function of p53 in cells overexpressing Skp2. Taken together, our results indicate that Skp2 controls p300-p53 signaling pathways in cancer cells, making Skp2 a potential molecular target for cancer therapy.