Ischemic stroke is closely associated with gut microbiota dysbiosis and intestinal barrier dysfunction. Prebiotic intervention could modulate the intestinal microbiota, thus considered a practical strategy for neurological disorders. Puerariae Lobatae Radix-resistant starch (PLR-RS) is a potential novel prebiotic; however, its role in ischemic stroke remains unknown. This study aimed to clarify the effects and underlying mechanisms of PLR-RS in ischemic stroke. Middle cerebral artery occlusion surgery was performed to establish a model of ischemic stroke in rats. After gavage for 14 days, PLR-RS attenuated ischemic stroke-induced brain impairment and gut barrier dysfunction. Moreover, PLR-RS rescued gut microbiota dysbiosis and enriched Akkermansia and Bifidobacterium. We transplanted the fecal microbiota from PLR-RS-treated rats into rats with ischemic stroke and found that the brain and colon damage were also ameliorated. Notably, we found that PLR-RS promoted the gut microbiota to produce a higher level of melatonin. Intriguingly, exogenous gavage of melatonin attenuated ischemic stroke injury. In particular, melatonin attenuated brain impairment via a positive co-occurrence pattern in the intestinal microecology. Specific beneficial bacteria served as leaders or keystone species to promoted gut homeostasis, such as Enterobacter, Bacteroidales_S24-7_group, Prevotella_9, Ruminococcaceae and Lachnospiraceae. Thus, this new underlying mechanism could explain that the therapeutic efficacy of PLR-RS on ischemic stroke at least partly attributed to gut microbiota-derived melatonin. In summary, improving intestinal microecology by prebiotic intervention and melatonin supplementation in the gut were found to be effective therapies for ischemic stroke.Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.