Sargassum fusiforme, widely consumed in Asian countries, has been proven to have various biological activities. However, the impacts and mechanisms of Sargassum fusiforme polysaccharides (SFPs) on intestinal bacterial infection are not yet fully understood. Our findings indicate that SFPs pretreatment ameliorates intestinal inflammation by reducing C. rodentium colonization, increasing colon length and levels of IL-10 and IL-22, decreasing IL-1β, IL-6, TNF-α, and IL-17 levels, inhibiting colonic crypt elongation and hyperplasia, and enhancing the intestinal mucosal barrier. The protective effects against intestinal bacterial infection are linked to enhanced clearance of C. rodentium and improvements in the intestinal mucosal barrier and C. rodentium-induced intestinal microbiota dysbiosis. Fecal microbiota transplantation experiments were conducted to evaluate the functional impact of microbiota induced by SFPs. The results suggest that intestinal microbiota modified by SFPs effectively countered C. rodentium infection. In addition, our study identified that miRNA-92a-3p is partially complementary to the 3'-UTR of the Notch1 gene, thereby repressing the Notch1-Hes1 signaling pathway and enhancing Muc2 secretion. Taken together, these findings reveal that SFPs protect mice from C. rodentium infection by activating the miR-92a-3p/Notch1-Hes1 regulatory axis driven by the intestinal microbiota, which stimulates Muc2 production to maintain intestinal barrier homeostasis.