The epithelium of the gastrointestinal (GI) tract is the largest mucosal surface of the body. This single layer of cells separates our tissues from the luminal content and is constantly exposed to microbial agents and dietary antigens. Epithelial barrier function is crucially promoted by immune cells named intraepithelial lymphocytes (IELs), these cells are tissue resident populations of predominantly T cells, including gd+ and ab+ T cells. IELs are distinct from conventional T cell subsets, their role in different compartments of the GI tract and potential to regulate cancer progression has not been studied in detail. We show that specific IEL populations are critical to limit tumour progression in mouse models of colon cancer and their presence is predictive of better prognosis in human disease. Using single cell RNA sequencing we show that colon IELs possess a unique molecular profile. This analysis led us to identify novel colon-specific transcriptional regulators of IEL development, response to commensal microbes and function in controlling tumour progression. Targeting these transcriptional regulators permitted selective ablation of colon IEL subsets without depleting IELs in other compartments of the GI tract. Our results open an exciting new avenue to exploit IEL diversity in GI diseases, specifically in colon cancer.