Background and purpose Severe diarrhoea, a common gastrointestinal manifestation of anticancer treatment with irinotecan, might involve single nucleotide polymorphisms (SNPs) of toll‐like receptors (TLRs), described as critical bacterial sensors in the gut. Here, colorectal cancer patients carrying missense TLR4 A896G (rs4986790) or C1,196T (rs4986791) SNPs and Tlr4 knockout (Tlr4−/−) mice were given irinotecan to investigate the severity of the induced diarrhoea. Experimental approach Forty‐six patients treated with irinotecan‐based regimens had diarrhoea severity analysed according to TLR4 genotypes. In the experimental setting, wild‐type (WT) or Tlr4−/− mice were given irinotecan (45 or 75 mg·kg−1, i.p.) or saline (3 ml·kg−1). Diarrhoea severity was evaluated by measuring intestinal injury and inflammatory markers expression after animals were killed. Key results All patients with TLR4 SNPs chemotherapy‐treated presented diarrhoea, whereas gastrointestinal toxicity was observed in 50% of the wild homozygous individuals. Mice injected with irinotecan presented systemic bacterial translocation and increased TLR4 immunostaining in the intestine. In line with the clinical findings, Tlr4 gene deficiency enhanced irinotecan‐related diarrhoea and TLR9 expression in mice. An increased myeloperoxidase activity and Il‐18 expression along with IL‐10 decreased production in Tlr4−/− mice also indicated an intensified intestinal damage and inflammatory response. Conclusion and implications TLR4 deficiency upregulates TLR9 expression and enhances intestinal damage and the severity of late‐onset diarrhoea during irinotecan‐based treatment. Identifying patients genetically predisposed to chemotherapy‐associated diarrhoea is a strategy toward precision medicine.