Murine double-minute clone 2 protein (MDM2) is an E3 ubiquitin ligase that regulates the turnover of several cellular factors including the p53 tumor suppressor protein. As part of the mechanism of p53 induction in response to DNA damage, a cluster of serine residues within the central acidic domain of MDM2 become hypophosphorylated, leading to attenuation of MDM2-mediated p53 destruction. In the present study, we identify the protein kinase CK1d as a major cellular activity that phosphorylates MDM2. Amino acid substitution, coupled with phosphopeptide analyses, indicates that several serine residues in the acidic domain, including Ser-240, Ser-242, and Ser-246, as well as Ser-383 in the C-terminal region, are phosphorylated by CK1d in vitro. We also show, through expression of a dominant negative mutant of CK1d or treatment of cells with IC261, a CK1d-selective inhibitor, that MDM2 is phosphorylated by CK1d in cultured cells. These data establish the identity of a key signaling molecule that promotes the phosphorylation of a major regulatory region in MDM2 under normal growth conditions.