Ischemic stroke is a major complication of type 2 diabetes mellitus (T2DM), significantly contributing to increased mortality in T2DM patients. Bone marrow mesenchymal stem cells (BMSCs), known for their multidirectional differentiation potential, have shown therapeutic potential in treating ischemic stroke associated with T2DM; however, the underlying mechanisms remain unclear. This study aimed to investigate the therapeutic effects and mechanisms of BMSCs in T2DM-related ischemic stroke. A T2DM rat model was established and subjected to transient middle cerebral artery occlusion (MCAO) to induce ischemic stroke. BMSCs were administered to evaluate their effects on body weight, blood glucose levels, modified neurological severity score (mNSS), infarct volume, and blood-brain barrier (BBB) integrity in T2DM-MCAO rats. RNA sequencing was performed on brain tissues from T2DM-MCAO rats before and after BMSCs treatment to identify differentially expressed genes (DEGs). Functional enrichment analysis, including Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, quantitative real-time PCR, and western blot, were conducted to explore the underlying mechanisms. The results demonstrated that T2DM-MCAO rats exhibited increased body weight, elevated blood glucose levels, higher mNSS scores, larger brain infarct volumes, and BBB disruption, all of which were partially ameliorated by BMSCs treatment. Furthermore, BMSCs downregulated the expression of TLR4 and reduced the protein levels of p65 and phosphorylated p65 (p-p65), which were upregulated in T2DM-MCAO rats. Overexpression of TLR4 partially reversed the beneficial effects of BMSCs on functional outcomes, infarct volume, and BBB integrity. In conclusion, this study demonstrates that BMSCs alleviate T2DM-related ischemic stroke by suppressing TLR4 expression and inhibiting the TLR4/NF-κB signaling pathway, suggesting a potential therapeutic target for T2DM-associated ischemic stroke.