Immune checkpoint inhibitors (CPIs) have revolutionised cancer treatment, with previously untreatable disease now amenable to potential cure. Combination regimens of anti-CTLA-4 and anti-PD-1 show enhanced efficacy but are prone to off-target immune-mediated tissue injury, particularly at the barrier surfaces. CPI-induced colitis is a common and serious complication. To probe the impact of immune checkpoints on intestinal homeostasis, mice were challenged with combination anti-CTLA-4/anti-PD-1 immunotherapy and manipulation of the intestinal microbiota. Colonic immune responses were profiled using bulk and single-cell RNA-sequencing and flow cytometry. CPI-colitis was dependent on the composition of the intestinal microbiota and was characterized by remodelling of mucosal lymphocytes with induction of polyfunctional lymphocyte responses characterized by increased expression of interferon-γ (IFNγ), other pro-inflammatory cytokines/chemokines (Il22, Il17a Ccl3, Ccl4 and Ccl9), cytotoxicity molecules (Gzmb, Gzma, Prf1, Nkg7) and the chemokine receptor Cxcr6. In comparison with mucosal lymphocytes in the steady state, polyfunctional lymphocytes from both CD4+ and CD8+ lineages upregulated costimulatory molecules and checkpoint molecules in CPI-colitis, indicating that these cells are tightly regulated. CPI-colitis was attenuated following depletion of effector lymphocytes or following blockade of the IL23/IFNγ axis. This study provides new mechanistic insights into CPI-colitis, identifying polyfunctional, cytotoxic lymphocytes as key mediators of disease. Therapeutic targeting of their effector response or regulatory networks, including the IL23/IFNγ axis likely holds the key to preventing and reversing CPI-colitis.