Transcriptional corepressors of the Groucho (Gro)/TLE family play important roles during a variety of developmental pathways, including neuronal differentiation. In particular, they act as negative regulators of neurogenesis, together with Hairy/Enhancer of split (Hes) DNA-binding proteins. The interaction with Hes1 leads to Gro/TLE hyperphosphorylation and increased transcription repression activity in mammalian cells, but the underlying molecular mechanisms are poorly characterized. We now show that Gro/TLE1 is phosphorylated in vivo by protein kinase CK2. This phosphorylation occurs at serine 239 within the conserved CcN domain present in all Gro/TLE family members. Mutation of serine 239 into alanine decreases Hes1-induced hyperphosphorylation of Gro/TLE1 and also reduces its nuclear association and transcription repression activity. We demonstrate further that Gro/TLE1 inhibits the transition of cortical neural progenitors into neurons and that its antineurogenic activity is inhibited by a serine-239-alanine mutation but not by a serine-239-glutamate mutation. These results suggest that CK2 phosphorylation of serine 239 of Gro/TLE1 is important for its function during neuronal differentiation.