BACKGROUND AND AIMS: Inhibiting gamma-secretase mediated Notch signaling has been explored as a potential treatment for Alzheimer's disease and cancer. However, clinical trials have revealed that this approach can lead to side effects, such as gut inflammation. Notch signaling has been shown to be a key mediator of intestinal epithelial homeostasis. We aimed to investigate the molecular mechanisms of gamma-secretase inhibition-associated colitis. METHODS: Mice and small intestinal organoids were treated with gamma-secretase inhibitors and analyzed for IEC differentiation and inflammation-associated markers using different molecular and histological approaches, along with transcriptomic and proteomic analyses. To evaluate the role of the microbiome in colitis development, mice undergoing pharmacological gamma-secretase inhibition were treated with antibiotics. Additionally, inflammatory bowel disease (IBD) patient samples and control samples were analyzed to assess the expression of Notch signaling pathway components in IECs. RESULTS: This study shows that pharmacological gamma-secretase inhibition induces inflammation in both the small and large intestine of mice, a phenotype that could be rescued upon microbiota depletion. Inhibiting the gamma-secretase induced structural disruption of the epithelium and inflammatory cytokine release. On a molecular level, epithelial organoids exhibited disrupted IEC differentiation and impaired proliferation, associated with defective Notch signaling. Finally, analysis of IBD patients revealed deregulation of Notch pathway components within IECs. CONCLUSIONS: In conclusion, systemic use of gamma-secretase inhibitors disrupts epithelial cell function by impairing IEC differentiation and triggering gut inflammation in mice. These findings should be considered when designing future therapeutic interventions involving gamma-secretase inhibitors.