© 2018, The Author(s). Delta ligands regulate Notch signaling in normal intestinal stem cells, while Jagged1 activates Notch in intestinal adenomas carrying active β-catenin. We used the ApcMin/+ mouse model, tumor spheroid cultures, and patient-derived orthoxenografts to address this divergent ligand-dependent Notch function and its implication in disease. We found that intestinal-specific Jag1 deletion or antibody targeting Jag1 prevents tumor initiation in mice. Addiction to Jag1 is concomitant with the absence of Manic Fringe (MFNG) in adenoma cells, and its ectopic expression reverts Jag1 dependence. In 239 human colorectal cancer patient samples, MFNG imposes a negative correlation between Jag1 and Notch, being high Jag1 in the absence of MFNG predictive of poor prognosis. Jag1 antibody treatment reduces patient-derived tumor orthoxenograft growth without affecting normal intestinal mucosa. Our data provide an explanation to Jag1 dependence in cancer, and reveal that Jag1–Notch1 interference provides therapeutic benefit in a subset of colorectal cancer and FAP syndrome patients.