Ebp1 is an ErbB3 binding phosphoprotein with pleiotropic effects. Overexpression of Ebp1 represses transcription of E2F1 responsive cell cycle regulated genes and inhibits cell growth. However, the effect of phosphorylation on Ebp1-mediated transcriptional repression and cell growth inhibition is currently unknown. In this study, we show that serine 363 (S363) of Ebp1 is phosphorylated in vivo. Although total Ebp1 is located in the nucleus, organelles and the cytoplasm, Ebp1 phosphorylated at S363 (Ebp1 pS363) is localized exclusively to the nucleus. Mutation of S363 to alanine did not change the subcellular localization of Ebp1. However, the S363A mutation significantly decreased the ability of Ebp1 to repress transcription and abrogated its ability to inhibit cell growth. We have previously shown that Ebp1 can bind the E2F1 promoter in vitro and in vivo as part of a protein complex and that Ebp1-transcriptional repression is mediated via its interaction with the co-repressors HDAC2 and mSin3a present in this complex. Although Ebp1 S363A interacted with an E2F1 promoter element, it did not bind HDAC2 and mSin3a. These results indicate the importance of S363 phosphorylation in the function of Ebp1.