Gut microbiome and plasma metabolome serve a role in the pathogenesis of ischemic stroke (IS). However, the relationship between the microbiota and metabolites remains unclear. This study aimed to reveal the specific asso-ciation between the microbiota and the metabolites in IS using integrated 16S rRNA gene sequencing and liquid chromatography-mass spectrometry (LC-MS) analysis. Male Sprague Dawley (SD) rats were divided into three groups: normal group (n = 8, Normal), model group (n = 9, IS), and sham-operated group (n = 8, Sham). Rats in the IS group were induced by middle cerebral artery occlusion (MCAO), and rats in the Sham group received an initial anesthesia and neck incision only. A neurological function test and 2,3,5-triphenyltetrazolium chloride (TTC) staining were used to assess the IS rat model. Then, the plasma samples were analyzed using untargeted LC-MS. The cecum samples were collected and analyzed using 16S rRNA sequencing. Pearson correlation analysis was performed to explore the association between the gut microbiota and the plasma metabolites. The 16S rRNA sequencing showed that the composition and diversity of the microbiota in the IS and control rats were significantly different. Compared with the Sham group, the abundance of the Firmicutes phylum was decreased, whereas Proteobacteria and Deferribacteres were increased in the IS group. Ruminococcus_sp_15975 and Lachnospiraceae_UCG_001 might be considered as biomarkers for the IS and Sham groups, respectively. LC-MS analysis revealed that many metabolites, such as L-leucine, L-valine, and L-phenylalanine, displayed different patterns between the IS and Sham groups. Pathway analysis indicated that these metabolites were mainly involved in mineral absorption and cholinergic synapse. Furthermore, integrated analysis correlated IS-related microbes with metabolites. For example, Proteobacteria were positively correlated with L-phenylalanine, while they were negatively correlated with eicosapentaenoic acid (EPA). Our results provided evidence of the relationship between the gut microbiome and plasma metabolome in IS, suggesting that these microflora-related metabolites might serve as potential diagnostic and therapeutic markers.