The transcription factor ER81 has been shown to be involved in ontogenesis and breast tumor formation. ER81 is activated by many signals through phosphorylation directly mediated by mitogen-activated protein kinases (MAPKs), but also by an unknown protein kinase(s). Here, mitogen- and stress-activated protein kinase 1 (MSK1), which itself is directly activated by distinct classes of MAPKs, is identified to regulate ER81 function. MSK1 expression enhances ER81-dependent transcription upon stimulation of especially the p38-MAPK pathway. Two serine residues in ER81 are phosphorylated by MSK1, and mutating these serine residues to alanines dramatically diminishes the ability of MSK1 to stimulate ER81. However, mutation of the MSK1 phosphorylation sites in ER81 does not completely abrogate the ability of MSK1 to activate ER81 function, suggesting that MSK1 may also target cofactors of ER81. Consistently, MSK1 interacts with two homologous coactivators of ER81, CBP and p300, and stimulates the transactivation domains of CBP. Thus, MSK1 may regulate ER81-dependent transcription via direct phosphorylation of ER81 as well as via stimulation of CBP/p300, which might be important for ER81's normal function and during mammary tumor formation.