Sepsis is one of the leading causes of mortality in severe systemic inflammatory syndrome. The endotoxin-induced inflammatory response has been linked to the development of sepsis. Rhein is a lipophilic anthraquinone isolated from Rheum rhabarbarum (rhubarb), which has a protective effect on intestinal damage in vivo. However, the underlying mechanism responsible for the protective effects of rhein remains to be elucidated. In the present study, mice were exposed to 20 mg/kg lipopolysaccharide (LPS), prior to being treated with either 100 mg/kg rhein or 0.3 mg/kg toll‑like receptor 4 (TLR4) signaling inhibitor TAK‑242. In the rhein‑treated mice, the colon length (cm) was extended and colon injury was attenuated. In addition, treatment with rhein significantly decreased the expression levels of the LPS‑induced inflammatory cytokines interleukin (IL)‑1β, IL‑6, IL‑8, and tumor necrosis factor‑α, in both the plasma and colon tissue. However, mice treated with TAK‑242 exhibited increased expression levels of IL‑10, as determined by ELISA and western blot analysis. In addition, immunohistochemistry and western blot analyses demonstrated that treatment with rhein was able to reduce TLR4 expression and inhibit nuclear factor‑κB (NF‑κB) phosphorylation in colon tissue. Furthermore, LPS induction was blocked by TAK‑242. These results demonstrate that the observed rhein‑attenuated inflammatory response during sepsis may be achieved via the TLR4 NF‑κB signaling pathway. In conclusion, the results of the present study provide a novel insight into the protective effects of rhein on LPS‑induced intestinal inflammation, and demonstrate that rhein may act as a beneficial therapeutic agent in the treatment of sepsis-induced intestinal damage.