Activation of the Wnt/β-catenin pathway is a critical step in the development of colorectal cancers. A key mediator of this activation is the recently described oncogene CDK8, which is amplified in a large number of colorectal tumors. CDK8 affects β-catenin activation by interaction of the CDK8 submodule of the mediator complex with β-catenin/TCF transcriptional complex, and by CDK8 interacting with and phosphorylating E2F1, which acts as a repressor of β-catenin/TCF transcriptional activity. The amino-acid residue in E2F1 that CDK8 phosphorylates and how this phosphorylation impacts E2F1 activity in general is not known. Here, we describe that CDK8 phosphorylates serine 375 in E2F1 both in vitro and in cells, and that phosphorylation of this residue is required for E2F1 interaction with CDK8, and that the phosphorylation is dependent on CDK8 kinase activity. The phosphorylation of S375 by CDK8 regulates E2F1 ability to repress transcription of β-catenin/TCF-dependent genes, as well as activation of E2F1-dependent genes. This regulation is due to inactivation of E2F1 transcriptional activation, and not to the interference of E2F1's ability to bind to E2F1-binding sites in various promoters or to interact with DP1.