This study investigated whether β-sitosterol has anti-allergic activity and explored its potential mechanism, using ovalbumin (OVA) allergic mouse model. Results indicated that supplementation with β-sitosterol at 5-20 mg kg-1 day-1 for 7 weeks alleviated allergic symptoms and intestinal inflammation, and reduced serum OVA-specific immunoglobulin (Ig) E, IgG and histamine levels in sensitized mice. β-Sitosterol enhanced physical and biochemical barrier in the intestinal epithelium by upregulating tight junction proteins (claudin-1, occludin, and ZO-1) expression and promoting the secretion of regenerating islet-derived protein IIIγ and secretory IgA in mucous layer. Furthermore, β-sitosterol administration increased the levels of interleukin 10 and transforming growth factor-β secreted by regulatory T cells, while reducing T helper 2 cell associated factor levels in intestinal lamina propria. Additionally, the alpha and beta diversity analysis revealed that the structure and diversity of the intestinal flora in the β-sitosterol group tended to be normalized compared with the model group, and the number of biomarkers was reduced from 32 to 7. Moreover, the altered composition of gut microbiota in allergic mice was also reversed by β-sitosterol supplementation, characterized by an increase in abundance of Lactobacillaceae and Bifidobacteriaceae and a decrease in abundance of Desulfovibrionaceae. Consequently, β-sitosterol may prevent FA by ameliorating intestinal barrier function and remodeling the gut microbiota.