Chemotherapy-induced intestinal mucositis (CIM) is a major dose-limiting side effect of many chemoagents, resulting in weight loss, diarrhea, and even death. The current treatments for CIM are palliative and have limited benefit. Interleukin-1 receptor antagonist is a natural antagonist of interleukin-1. Our previous studies showed the protective effect of recombinant human interleukin-1 receptor antagonist (rhIL-1Ra) on the intestine in mice after 5-fluorouracil chemotherapy. In this study, we further evaluated rhIL-1Ra in the treatment of CIM induced by different chemoagents and their combination. Normal as well as tumor-bearing mice were administered oxaliplatin (L-OHP), 5-fluorouracil, or their combination to induce intestinal mucositis and mortality. rhIL-1Ra administered after the chemotherapy, but not after the onset of diarrhea, significantly improved mouse survival, attenuated body weight loss, and reduced the incidence, severity, and duration of diarrhea. Histological examination showed that rhIL-1Ra-treated mice had a relatively intact mucosa structure, more proliferating crypt cells, and higher acid mucin content than the vehicle-treated mice. rhIL-1Ra suppressed crypt apoptosis by reducing the levels of proapoptotic proteins in wild-type, but not in IL-1RI or p53 mice. In addition, rhIL-1Ra was as effective as octreotide acetate in the treatment of chemotherapy-induced diarrhea, but with the advantage of reducing the epithelial apoptosis, the major cause of CIM. Importantly, the tumor sensitivity to chemotherapy was not affected by rhIL-1Ra. Thus, our data strongly suggest that rhIL-1Ra may be useful for the treatment of intestinal mucositis and improving the quality of life for cancer patients on chemotherapy.