Chemotherapies are known often to induce severe gastrointestinal tract toxicity but the underlying mechanism remains unclear. This study considers the widely applied cytotoxic agent irinotecan (CPT-11) as a representative agent and demonstrates that treatment induces massive release of double-strand DNA from the intestine that accounts for the dose-limiting intestinal toxicity of the compound. Specifically,“self-DNA” released through exosome secretion enters the cytosol of innate immune cells and activates the AIM2 (absent in melanoma 2) inflammasome. This leads to mature IL-1β and IL-18 secretion and induces intestinal mucositis and late-onset diarrhoea. Interestingly, abrogation of AIM2 signalling, either in AIM2-deficient mice or by a pharmacological inhibitor such as thalidomide, significantly reduces the incidence of drug-induced diarrhoea without affecting the anticancer efficacy of CPT-11. These findings provide mechanistic insights into how chemotherapy triggers innate immune responses causing intestinal toxicity, and reveal new chemotherapy regimens that maintain anti-tumour effects but circumvent the associated adverse inflammatory response.