Most sporadic colorectal cancers are initiated by activating Wnt pathway mutations, characterized by the stabilization of β-catenin and constitutive transcription by the β-catenin/T cell factor-4 (Tcf-4) complex^,. EphB guidance receptors are Tcf4 target genes that control intestinal epithelial architecture through repulsive interactions with Ephrin-B ligands^,. Here we show that, although Wnt signalling remains constitutively active, most human colorectal cancers lose expression of EphB at the adenoma–carcinoma transition. Loss of EphB expression strongly correlates with degree of malignancy. Furthermore, reduction of EphB activity accelerates tumorigenesis in the colon and rectum of Apc^Min/+ mice, and results in the formation of aggressive adenocarcinomas. Our data demonstrate that loss of EphB expression represents a critical step in colorectal cancer progression.