Background: Ischemic stroke (IS) continues to be the foremost cause of mortality and disability worldwide. Ginkgolide B (GB), an originally natural terpene lactone, is widely employed in the treatment of cerebrovascular disease. Nevertheless, a comprehensive understanding of the precise role and underlying mechanisms of GB in white matter injury after IS necessitates further elucidation. The objective of the present study was to assess the impact of GB on white matter injury and to analyze alterations in the types and gene expression of neuroglial cells using single-cell RNA sequencing (scRNA-seq). Methods: Ninety-four male C57BL/6 mice were categorized into four groups: (1) Sham control (n = 21); (2) middle cerebral artery occlusion (MCAO) mice receiving PBS vehicle (MCAO+PBS) group (n = 29); (3) MCAO mice subjected to a 1-week-long GB treatment (MCAO+GB1w) group (n = 13); (4) MCAO mice undergoing a 2-week-long GB treatment (MCAO+GB2w) group (n = 31). GB or PBS was administered to the mice following ischemic cerebral injury induced by middle cerebral artery occlusion (MCAO). Neurobehavioral state was assessed using foot fault (FF) and rotarod (RR) tests. White matter injury and microglial cell subtypes were determined through immunofluorescence. ScRNA-seq was employed to illustrate the crucial biological processes and gene expressions influenced by GB in white matter injury after MCAO. Results: Our findings indicated a significant prolongation in the latency to fall during RR test and a decrease in the FF rate for both forelimb and hindlimb in the MCAO+GB2w group compared to the MCAO+PBS group. The demyelinating damage to white matter in the MCAO+GB2w group was notably milder than that in the MCAO+PBS group (p < 0.05), as evidenced by a significantly increased MBP/SMI-32 ratio. Immunofluorescence analysis revealed an elevated proportion of M2 microglial cells and a decreased level of M1 microglial cells in the MCAO+GB2w group compared to the MCAO+PBS group on the 14th day, 21st day, and 28th day (p < 0.05). ScRNA-seq findings demonstrated that GB provides protection against ischemia/reperfusion (I/R)-induced white matter injury by upregulating critical processes such as myelin sheath reproduction and oligodendrocyte maturation. Gene Ontology (GO) enrichment and analysis of differentially expressed genes (DEGs) revealed a significant upregulation of proteolipid protein 1 (PLP1), alphaB-Crystallin (CRYAB), ß-tubulin 4A (TUBB4A), constitutive androstane receptor 2 (CAR2), and ATPase Na⁺/K⁺ transporting subunit beta 2 (ATP1B2) in the myelin sheath pathway of microglial cells. Similarly, echinoderm microtubule-associated protein-like 1 (EML1), Heat shock proteins 72 (HSPA1A), signal peptide peptidase-like 2b (SPPL2B), and alpha 1A (TUBA1A) were significantly upregulated in oligodendrocytes (p < 0.05). Cell trajectory analysis indicated that oligodendrocytes underwent induction towards more mature subclusters following GB administration. Conclusions: GB has the potential to alleviate white matter injury and neurobehavioral dysfunction following MCAO by increasing the proportion of M2 microglial cells, decreasing M1 microglial cells, and promoting oligodendrocyte maturation to enhance myelination.