Background: Anthraquinone glycosides extracted from rhubarb have been proven to have significant therapeutic effects on ischaemic stroke. It is well known that anthraquinone glycosides are not easily absorb. Thus, how can rhubarb anthraquinone glycosides (RAGs) exert protective effects on the brain? Is this protective effect related to interactions between RAGs and intestinal flora? Methods: The model used in this study was established by middle cerebral artery occlusion (MCAO) and reperfusion. Twenty-seven adult male Sprague–Dawley (SD) rats were randomly divided into 3 groups: the normal group (A) (non-MCAO + 0.5% sodium carboxymethyl cellulose (CMC-Na)), model group (B) (MCAO + 0.5% CMC-Na) and medicine group (C) (MCAO + RAGs (15 mg/(kg day)). The rats were fed by gavage once a day for 7 days. Fresh faeces were collected from the normal group to prepare the intestinal flora incubation liquid. Add RAGs, detect the RAGs and the corresponding anthraquinone aglycones by HPLC–UV at different time points. On the 8th day, the rats were euthanized, and the colonic contents were collected and analysed by high-throughput sequencing. In addition, 12 adult male SD rats were randomly divided into 2 groups: the normal group (D) (non-MCAO + RAGs (15 mg/(kg day)) and model group (E) (MCAO + RAGs (15 mg/(kg day)). The rats were fed by gavage immediately after reperfusion. Blood was collected from the orbital venous plexus, and the RAGs and anthraquinone aglycones were detected by HPLC–UV. Results: The abundance and diversity of the intestinal flora in rats decreased after cerebral ischaemia–reperfusion injury (CIRI). RAGs could effectively improve the abundance of the intestinal flora. In addition, in vitro metabolism studies showed that RAGs were converted into anthraquinone aglycones by intestinal flora. In the in vivo metabolism studies, RAGs could not be detected in the plasma; in contrast, the corresponding anthraquinone aglycones could be detected. Absorption of RAGs may be inhibited in rats with CIRI. Conclusions: CIRI may lead to intestinal flora disorder in rats, and after the administration of RAGs, the abundance of intestinal flora can be improved. RAGs can be metabolized into their corresponding anthraquinone aglycones by intestinal flora so that they can be absorbed into the blood.