Irinotecan (CPT-11) is used to treat various cancers but side effects such as delayed diarrhea restrict its use. Darunavir (DRV) is an antiretroviral drug used to treat and prevent human immunodeficiency virus/acquired immune deficiency syndrome (HIV/AIDS), but whether DRV is protective against CPT-11-induced intestinal toxicity is unclear. An CPT-11-induced intestinal toxicity model was produced using uninterrupted CPT-11 (ip) for 4 d in mice. Enzyme-linked immuno sorbent assay (ELISA), fecal occult blood test (FOBT), Western blot, histopathological evaluation, and immunohistochemistry staining assays were used to document toxicity. DRV treatment attenuated CPT-11-induced intestinal toxicity via decreasing fecal occult blood and mitigating delayed-onset diarrhea, as well as reducing weight loss, reduced food intake, and pathomorphologic changes without inhibiting β-glucuronidase (β-GLU) activity. The high mobility group box-1 protein (HMGB1)-toll-like receptor 4 (TLR4) pathway induced inflammation and tight junction protein (occludin and zonular occluden-1) reduction in the colon was inhibited by DRV. Hepatotoxicity induced by CPT-11 was diminished after treatment with DRV, and activation of the NOD-like receptor 3 inflammasome (NLRP3) was prevented in colon tissue. In addition, DRV didn’t reduce the concentration of CPT-11 and 7-ethyl-10-hydroxycamptothecin (SN-38) in plasma at the same dose of irinotecan with DRV. DRV has anti-inflammatory and intestinal-protective properties and may be used to manage CPT-11-induced intestinal toxicity.