Abstract: Ischemic stroke (IS) remains a major contributor to global disability and mortality. Astragalus polysaccharide (ASP), a naturally active component derived from Astragalus membranaceus, exhibits therapeutic potential against IS. However, their mechanism against IS via the microbiota-gut-brain axis remains unclear. Our study aimed to evaluate the mechanism of ASP against IS by middle cerebral artery occlusion (MCAO)-induced animal models combined with antibiotics (ABX) and fecal microbiota transplantation (FMT) experiments. In MCAO mice, our results showed that ASP significantly attenuated brain injury and intestinal barrier dysfunction. Transcriptomics, network pharmacology, and western blot identified LPS-TLR4-MAPK pathway as a key regulatory pathway in the regulation of IS-induced intestinal barrier dysfunction by ASP. Metagenomics and metabolomics indicated that ASP modulates SCFA-producing and anti-inflammatory bacterial genera (g_Anaerobutyricum and g_Caproiciproducens). Critically, ABX and FMT experiments confirmed that ASP’s neuroprotective effects in MCAO mice receiving gut microbiota from IS patients, with this therapeutic benefit being microbiota-dependent. Additionally, LPS levels were upregulated in clinical patients with IS. In conclusion, our findings indicated that ASP alleviates IS-induced brain injury via the microbiota-gut-brain axis.