Cancer stem cells (CSC) have attracted attention as therapeutic targets; however, CSC-targeting therapy may disrupt normal tissue homeostasis because many CSC molecules are also expressed by normal stem cells (NSC). Here, we demonstrate that NSC-specific and CSC-specific roles of the stem cell transcription factor Hes1 in the intestine enable the feasibility of a specific cancer therapy. Hes1 expression was upregulated in NSCs and intestinal tumors. Lineage-tracing experiments in adult mouse intestine revealed that Hes1 deletion in Lgr5(+) or Bmi1(+) NSCs resulted in loss of self-renewal but did not perturb homeostasis. Furthermore, in Lgr5(+) NSC, deletion of Hes1 and beta-catenin stabilization limited tumor formation and prolonged host survival. Notably, in Lgr5(+) or Dclk1(+) tumor stem cells derived from established intestinal tumors, Hes1 deletion triggered immediate apoptosis, reducing tumor burden. Our results show how Hes1 plays different roles in NSCs and CSCs, in which Hes1 disruption leads to tumor regression without perturbing normal stem cell homeostasis, preclinically validating Hes1 as a cancer therapeutic target. (C) 2017 AACR.