Abstract Notch-Hes pathway plays a crucial role in the differentiation, proliferation, and tumorigenesis of the intestine. We previously reported that prenatal deletion of Hes1 reduces intestinal tumor proliferation; however, postnatal deletion of Hes1, which recapitulates Hes1-targeted tumor therapy, has not been thoroughly investigated. Here, we elucidated the novel role of Hes1 in normal stem cells (NSCs) and tumor stem cells (TSCs) of the adult intestine. First, we deleted Hes1 in Lgr5-expressing or Bmi1-expressing NSCs and performed lineage tracing using Lgr5CreERT2/+; Hes1flox/flox; Rosa26LacZ/+ and Bmi1CreER/+; Hes1flox/flox; Rosa26LacZ/+ mice. After Hes1 deletion, Lgr5-expressing or Bmi1-expressing NSCs gave rise to progeny cells, but could not self-renew. However, Hes1 deletion in NSCs did not disrupt intestinal homeostasis, because Hes1-positive NSCs were newly generated soon after Hes1-deleted NSCs were depleted. Next, we stabilized beta-catenin for intestinal tumor formation and deleted Hes1 in Lgr5-expressing cells using Lgr5CreERT2/+; Ctnnb1lox(ex3)/+; Hes1flox/flox mice. Tumor formation was dramatically suppressed, and the animal survival was significantly improved, suggesting that Hes1 is required for the initiation of intestinal tumor formation. Finally, we deleted Hes1 in Lgr5-expressing TSCs of established intestinal tumors using Lgr5CreERT2/+; Hes1flox/flox; ApcMin/+ mice. Of note, Hes1 deletion in TSCs of established intestinal tumors induced immediate apoptosis of TSCs and significantly reduced the number of tumors. qRT-PCR analysis of sorted Lgr5-expressing cells one day after Hes1 deletion revealed upregulation of pro-apoptotic genes and downregulation of anti-apoptotic genes in Hes1-deleted TSCs, but not in Hes1-deleted NSCs. We also deleted Hes1 in Dclk1-expressing TSCs of established intestinal tumors, and confirmed that, irrespective of stem cell markers analyzed, Hes1 deletion in TSCs leads to tumor regression by inducing immediate apoptosis in TSCs. Taken together, Hes1 is required in different ways for the maintenance of NSCs and TSCs: Hes1 deletion in NSCs does not perturb homeostasis, whereas Hes1 deletion in TSCs leads to tumor regression. Furthermore, Hes1 is necessary for the initiation of intestinal tumor formation. Therefore, we propose Hes1 is a novel tumor-specific therapeutic target. Citation Format: Norihiro Goto, Akihisa Fukuda, Tsutomu Chiba, Hiroshi Seno. The role of Hes1 in the normal and tumor stem cells of the intestine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4771. doi:10.1158/1538-7445.AM2017-4771