Ischemic brain injury (IBI) is characterized by high morbidity, disability and mortality rates; however, it lacks effective clinical treatments. Mesenchymal stem cells (MSCs), as pluripotent stem cells with self‑renewal capacity and multilineage differentiation potential, have emerged as a promising therapeutic strategy for neurological disorders. In the present study, in vitro experiments were performed using the Wnt signaling agonist Wnt3a and the B lymphoma Mo‑MLV insertion region 1 homolog (Bmi1) small molecule inhibitor PTC209 to treat MSCs, and the roles and regulatory mechanisms of the Bmi1 and Wnt3a‑RhoA signaling pathways on the neural differentiation of MSCs were explored by MTT assay, immunofluorescence analysis and western blotting. In vivo experiments were also performed by establishing a rat model of middle cerebral artery occlusion (MCAO), transplanting different MSCs into the rat brain tissues after in vitro labeling, and comparing ischemic brain damage in each group of rats by Neurological Severity Score scoring, grasp assay, triphenyltetrazolium chloride staining, hematoxylin and eosin staining, and assessing neurological recovery via immunofluorescence and western blot analysis. The in vivo study aimed to assess the roles of the Bmi1 and Wnt3a‑RhoA signaling pathways in brain injury repair in MCAO rats and the mechanism. Specifically, recombinant Wnt3a cytokine was administered to upregulate the Wnt3a‑RhoA pathway, whereas the small‑molecule inhibitor PTC209 was utilized to suppress Bmi1 expression. The findings suggested that Bmi1 modulates the neural differentiation of MSCs through its regulatory effects on Wnt3a and RhoA expression, thereby influencing the reparative potential of MSCs in ischemic brain tissue. These findings highlight the therapeutic relevance of targeting Wnt3a‑RhoA activation and Bmi1 inhibition in MSC‑based interventions for IBI.