A 5-fluorouracil (5-FU) is used for cancer treatment despite its cytotoxic sequelae on healthy cells, especially the rapid proliferating ones. Intestinal mucositis is one of the most frequent chemotherapeutic debilitating sequelae. Rhubarb (Rh), an ancient herb, is known for its curing effect on gastrointestinal complications. This study aims to detect the role of aquaporin-4 (AQP-4), tumour necrosis factor-α (TNF-α), nuclear factor-kappa B (NF-κB), and matrix metalloproteinase-9 (MMP-9) in 5-FU-induced ileal histological and biochemical changes and the potential therapeutic effect of Rh water extract on these changes in rats. A 45 rats were divided into 3 groups; control, 5-FU (single intraperitoneal injection of 150 mg/kg/rat) and Rh-treated (oral 20 mg/kg/day/rat for 8 days). The change in animals’ weight, incidence of diarrhoea and AQP-4 and TNF-α values in ileal homogenates were measured. Ileal sections were subjected to hematoxylin and eosin stain, periodic acid Schiff (PAS)-reaction and MMP-9, NF-κB and AQP-4 immunohistochemical staining. A 5-FU group revealed marked ileal mucosal damage associated with a significant decrease in the mean body weight, AQP-4 level and area percent of PAS and AQP-4 positive reaction. Significant increase in the mean incidence of diarrhoea, TNF-α value and area percent of MMP-9 and NF-κB was detected. These changes were significantly corrected with Rh administration. A 5-FU resulted in severe ileal mucositis through TNF-α, NF-κB, MMP-9, and AQP-4 disturbances. Rh treatment was highly effective in preventing such mucositis.