Irinotecan treatment of colorectal cancers results in high-grade intestinal mucositis in a large proportion of patients. The mechanisms behind irinotecan-induced mucosal injury, however, have yet to be fully explained. The aim of this study was to investigate the role of the p53 protein in the onset of intestinal damage following irinotecan treatment in two different settings. IEC-6 and FHs 74 intestinal cell lines were treated with irinotecan with and without a temporary p53 inhibitor, pifithrin-alpha, and examined for changes in proliferation and survival along with expression of p53 and related proteins. Forty tumour-bearing rats also underwent irinotecan treatment with and without pifithrin-alpha, and the effects on intestinal morphology, gene expression, apoptosis and other toxicities were assessed. Irinotecan caused a dose-dependent reduction in cell viability that was not prevented by pifithrin-alpha in either cell line. Rats responded to irinotecan with diarrhoea, weight loss, histopathological changes to the small and large intestine, increased crypt apoptosis, and a mild inflammatory response. Pifithrin-alpha reduced severity and duration of intestinal apoptosis; however, it did not significantly affect other parameters including p53 expression. Temporary inhibition of p53 activation does not markedly prevent intestinal cell death or mucositis following irinotecan treatment. Irinotecan may act through upregulation of proapoptotic proteins Bax and Bak to induce cell death.