Background and Purpose: 5-Fluorouracil (5-FU)-based chemotherapy is the first-line chemotherapeutic agent for colorectal cancer. However, 5-FU–induced intestinal mucositis (FUIIM) is a common adverse effect that severely impairs drug tolerance and results in poor patient health. Experimental Approach: Neutrophil elastase (NE) overexpression contributes to FUIIM via abnormal inflammatory responses, microbiota imbalance, and tissue damage. Therefore, restoring NE homeostasis could prevent or improve FUIIM. Key Results: This study shows that treatment with the specific NE inhibitor MPH966 (7.5 mg/kg; p.o.) significantly reversed 5-FU–induced losses in body weight; reversed villus atrophy; significantly suppressed myeloperoxidase, NE, and proteinase 3 activity; and reduced pro-inflammatory cytokine levels in a mouse model of FUIIM. In addition, MPH966 prevented 5-FU–induced intestinal barrier dysfunction, as was indicated by modulated expression of the tight junction proteins zonula occludin-1 and occludin. MPH966 also reversed 5-FU–induced changes in gut microbiota diversity and abundances, specifically the Firmicutes-to-Bacteroidetes ratio; Muribaculaceae, Ruminococcaceae, and Eggerthellaceae abundances at the family level; and Candidatus Arthromitus abundance at the genus level. Conclusion and Implications: These data indicate that NE inhibitors are a potential treatment candidate to alleviate FUIIM by regulating abnormal inflammatory responses, intestinal barrier dysfunction, and gut microbiota imbalance.