Mitogen- and stress-activated kinase 1 (MSK1) is a chromatin kinase that facilitates activator-dependent transcription by altering chromatin structure through histone H3 phosphorylation. The kinase activity of MSK1 is activated by intramolecular autophosphorylation, which is initially triggered by the activation of upstream mitogen-activated protein kinases (MAPKs), such as p38 and ERK1/2. MSK1 has been implicated in the expression of p21, a p53 target gene; however, the precise connection between MSK1 and p53 has not been clearly elucidated. Here, using in vitro and cell-based transcription assays, we show that MSK1 functions as a transcriptional coactivator of p53 in p21 expression, an action associated with MAPK-dependent phosphorylation of MSK1 and elevated kinase activity. Of special significance, we show that MSK1 directly interacts with p53 and is recruited to the p21 promoter, where it phosphorylates histone H3 in a p53-dependent manner. In addition, phosphomimetic mutant analysis demonstrated that negative charges in the hydrophobic motif are critical for serine 212 phosphorylation in the N-terminal kinase domain, which renders MSK1 competent for histone kinase activity. These studies suggest that MSK1 acts through a direct interaction with p53 to function as a transcriptional coactivator and that MSK1 activation by upstream MAPK signaling is important for efficient p21 gene expression.