Intestinal mucosal barrier damage is closely associated with the development of several intestinal inflammatory diseases. Isoquercitrin (IQ) is a natural flavonoid compound derived from plants, which exhibits high antioxidant and anti-inflammatory activity with minimal side effects in humans. Therefore, it shows great potential for preventing and treating intestinal mucosal barrier damage. This study aims to investigate the ameliorative effect and mechanism of IQ on lipopolysaccharide (LPS)-induced intestinal mucosal barrier damage in mice. The mice were treated with IQ for 7 days and then injected with LPS to induce intestinal mucosal barrier damage. The results revealed that IQ treatment alleviated LPS-induced intestinal mucosal barrier damage in mice, which can be evidenced by the improvements in intestinal morphology and the promotion of expression in intestinal tight junctions (ZO-1, Claudin-1, and Occludin), as well as MUC2 mucin. IQ also attenuated intestinal inflammatory responses by inhibiting the TLR4/MyD88/NF-κB signaling pathway and reducing the expression and plasma levels of IL-6, IL-1β, and TNF-α. Furthermore, IQ significantly increased the relative abundance of beneficial bacteria, including Dubosiella, Akkermansia muciniphila and Faecalibaculum rodentium, while suppressing the growth of harmful bacteria such as Mucispirillum schaedleri in the intestinal flora of mice. Consequently, IQ can alleviate the LPS-induced intestinal mucosal barrier damage in mice by inhibiting the TLR4/MyD88/NF-κB signaling pathway and modulating the intestinal flora.