Background: Stroke is a major cause of morbidity and mortality worldwide. After cerebral ischemia, peripheral immune cells infiltrate the brain and elicit an inflammatory response. However, it is not clear when and how these peripheral immune cells affect the central inflammatory response, and whether interventions that target these processes can alleviate ischemia-reperfusion (I/R) injury. Methods: Single-cell transcriptomic sequencing and bioinformatics analysis were performed on peripheral blood of mice at different times after I/R to analyze the key molecule of cell subsets. Then, the expression pattern of this molecule was determined through various biological experiments, including quantitative RT-PCR, western blot, ELISA, and in situ hybridization. Next, the function of this molecule was assessed using knockout mice and the corresponding inhibitor. Results: Single-cell transcriptomic sequencing revealed that peripheral monocyte subpopulations increased significantly after I/R. Cathepsin S (Ctss)was identified as a key molecule regulating monocyte activation by pseudotime trajectory analysis and gene function analysis. Next, Cathepsin S was confirmed to be expressed in monocytes with the highest expression level 3 days after I/R. Infarct size (p < 0.05), neurological function scores (p < 0.05), and apoptosis and vascular leakage rates were significantly reduced after Ctss knockout. In addition, CTSS destroyed the blood-brain barrier (BBB) by binding to junctional adhesion molecule (JAM) family proteins to cause their degradation. Conclusions: Cathepsin S inhibition attenuated cerebral I/R injury; therefore, cathepsin S can be used as a novel target for drug intervention after stroke. Keywords: Blood–brain barrier(4); Cathepsin S(1); Junctional adhesion molecule(3); Neuroinflammation(2); Stroke(5).