Background: The Atonal Homolog 1 (Atoh1 or Math1) transcription factor is required for intestinal secretory (goblet, Paneth, enteroendocrine) cell differentiation. Notch/γ secretase inhibitors (GSIs) block proliferation and induce secretory cell differentiation in the intestine. Here, we genetically determined whether Atoh1 mediates the effects of GSIs in both normal and cancerous intestinal epithelia. Methods: Mice with intestine-specific Atoh1 mutation (Atoh1Δintestine), APCmin mutation, double mutation [Atoh1Δintestine; APCmin], and littermate controls, were treated with GSI or vehicle. Colorectal cancer (CRC) cell lines were treated with GSI or vehicle, and with shRNAs to reduce ATOH1. Differentiation and homeostasis was assessed by quantitative gene expression and histologic analysis. Results: Unlike wild type controls, GSIs failed to induce secretory cell differentiation or apoptosis, or decrease proliferation of Atoh1-null progenitor cells. Similarly, GSI treatment of APCmin adenomas decreased proliferation and increased secretory cells in an Atoh1-dependent fashion. In CRC cells treated with GSI, ATOH1 was inversely correlated with proliferation and was required for secretory cell gene expression. Conclusions:We find that ATOH1 is critical for all effects of GSIs in intestinal crypts and adenomas, suggesting that Notch has no unique function in intestinal progenitors and cancer cells other than regulating appropriate ATOH1 expression. Reducing or circumventing ATOH1may mitigate intestinal toxicity from systemic GSI therapy for non-intestinal diseases. Among gastrointestinal malignancies, ATOH1 is a critical mediator of GSI effects, and ATOH1 expression status may predict response to GSI. We suggest that only the subset of CRCs which retain ATOH1 expression will respond to GSI therapy.