Intestinal stem cells (ISCs) maintain regenerative capacity of the intestinal epithelium. Their function and activity are regulated by transcriptional changes, yet how such changes are coordinated at the genomic level remains unclear. The Cohesin complex regulates transcription globally by generating topologically-associated DNA domains (TADs) that link promotor regions with distant enhancers. We show here that the Cohesin complex prevents premature differentiation of ISCs into enterocytes (ECs). Depletion of the Cohesin subunit Rad21 and the loading factor Nipped-B triggers an ISC to EC differentiation program that is independent of Notch signaling, but can be rescued by over-expression of the ISC-specific escargot (esg) transcription factor. Using damID and transcriptomic analysis, we find that Cohesin regulates Esg binding to promoters of differentiation genes, including a group of Notch target genes involved in ISC differentiation. We propose that Cohesin ensures efficient Esg-dependent gene repression to maintain stemness and intestinal homeostasis.