Inhibition of Notch signaling is effective in inhibiting colon tumorigenesis, but targeting specific components of the pathway may provide more effective strategies. Here we show that the expression of Jagged1, a ligand for canonical Notch signaling, was restricted to enteroendocrine cells or undetectable in the mucosa of the human small and large intestine, respectively. In contrast, increased expression characterized half of human colon tumors, although not all tumors with elevated Wnt signaling displayed elevated Jagged1. Increased Jagged1 was also present in intestinal tumors of Apc(1638N/+) and Apc(Min/+) mice, but to a higher level and more frequently in the former, and in 90% of mouse tumors Notch signaling was elevated when Jagged1 was elevated. In the human HT29Cl16E colonic carcinoma cell line, induction of goblet cell differentiation by contact inhibition of growth depended on the loss of Jagged1-mediated Notch activation, with signaling through Notch1 and Notch2 acting redundantly. Therefore, targeting of Jagged1 could be effective in downregulating Notch signaling in a subset of tumors, but may avoid the limiting gastrointestinal toxicity caused by pharmacological inhibition of Notch signaling.