CITED1 is the founding member of the CITED family of cofactors that are involved in regulating a wide variety of CBP/p300-dependent transcriptional responses. In the present study, we show that the phosphorylation status of CITED1 changes during the cell cycle and affects its transcriptional cofactor activity. Tryptic mapping and mutagenesis studies identified five phosphorylated serine residues in CITED1. Phosphorylation of these residues did not affect CRM1-dependent nuclear export, but did decrease CITED1 binding to p300 and inhibited CITED1-dependent transactivation of Smad4 and p300. These results suggest that CITED1 functions as a cell cycle-dependent transcriptional cofactor whose activity is regulated by phosphorylation.