Abstract: The intestinal barrier, as the primary interface between the body and the external environment, plays a crucial role in sepsis. Methylene blue, with its anti-inflammatory, antioxidant, and microcirculatory-improving properties, has been widely used in the treatment of sepsis. However, whether it can improve intestinal barrier function in sepsis and its underlying mechanisms remain unclear. In the present study, a sepsis mouse model was established via intraperitoneal injection of lipopolysaccharide (LPS). The effects of methylene blue (5-20 mg·kg^-1) on inflammation in septic mice were observed using HE staining, ELISA, and RT-PCR. Changes in intestinal barrier function in the jejunum and colon tissues were analyzed via Evans blue staining, immunofluorescence, and RT-PCR. Additionally, the effects of methylene blue (40 μg·mL^-1) on NF-κB and RhoA/ROCK signaling pathways in LPS-stimulated macrophages (RAW264.7) and intestinal epithelial cells (Caco-2) were evaluated. The animal study was approved by the Animal Ethics Committee of Southwest University (Approval No.: IACUC-20241125-04). Results showed that methylene blue (5-20 mg·kg^-1) significantly reduced the levels of inflammatory cytokines in plasma and intestinal tissues of septic mice and upregulated the expression of tight junction proteins in the jejunum and colon. In vitro experiments confirmed that methylene blue (40 μg·mL^-1) inhibited the increased secretion of inflammatory cytokines in LPS-stimulated RAW264.7 cells, a mechanism associated with NF-κB pathway inhibition. Methylene blue (40 μg·mL^-1) also reversed the LPS-induced decrease in transmembrane electrical resistance and cell migration in Caco-2 cells, a mechanism related to RhoA/ROCK pathway inhibition. In summary, the present study demonstrated that methylene blue can improve intestinal inflammation by inhibiting the NF-κB pathway and enhance intestinal barrier function via the RhoA/ROCK pathway, ultimately alleviating the progression of sepsis.