Abstract Metabolic dysfunction-associated steatohepatitis (MASH) has become a serious public health problem, posing an increasingly dangerous threat to human health owing to its increasing prevalence and accompanying intra- and extrahepatic adverse outcomes. Rifaximin is considered to have therapeutic potential for MASH; however, its efficacy remains controversial. Our study aimed to observe the ameliorative effects of rifaximin and explore its possible mechanisms at the cellular level. 1. 42 male C57BL/6J mice were divided into 3 groups, the CON group and MCD group were fed with normal feed and MCD feed for 12 weeks respectively, and the MCD + RFX group was treated with rifaximin by gavage for 4 weeks on the basis of MCD feed. Hematoxylin-eosin staining, Sirius red staining and immunohistochemical staining were used to observe the histopathological changes of liver and intestine. Differences in liver transaminases, inflammatory factors, fibrosis indexes and intestinal tight junction proteins were compared among the 3 groups of mice. 2. A MASH cell model was constructed by inducing HepG2 cells with free fatty acids to observe the effects of rifaximin on MASH in vitro. In addition, the effects of rifaximin on TLR4/NF-κB signaling pathway were explored by applying TLR4 agonist LPS and TLR4 inhibitor TAK-242. Hepatic histopathology was significantly improved in MASH mice after rifaximin treatment, and their serum alanine aminotransferase and aspartate aminotransferase levels were (72.72 ± 5.68) U/L and (222.8 ± 11.22) U/L, respectively, which were significantly lower than those in the MCD group [(293.3 ± 10.69) U/L and (414.1 ± 36.29) U/L, P