Background Vascular disease in diabetes, for example, stroke, presents a significant public health burden. Recently, the dipeptidyl peptidase 4 (DPP-4) inhibitor linagliptin has been found to counteract stroke among diabetic patients, showing great promise in drug repurposing and indication expansion. However, the molecular basis of this protection mechanism remains unknown. Methods The expression and localization of DPP-4 in rat brain microvascular endothelial cells (rBMVECs) were assessed with immunofluorescent staining and Western blotting. The effects of DPP-4 inhibitors on cell proliferation and migration of rBMVECs were determined using MTT and transwell assays, separately. The influence of DPP-4 inhibition on the expression of molecular markers (eg, VEGF, eNOS, HIF-1 alpha. SIRT1) was examined at both mRNA and protein levels with qRT-PCR and Western blotting, individually. Results DPP-4 inhibitors (40 nmol/L linagliptin, 30 mu mol/L berberine) offer protection from hypoxia/high glucose induced impairments in the proliferation and migration of rBMVECs. Treatment with DPP-4 inhibitors counteracted the attenuating effects of hypoxic/high-glucose conditions on the expression of VEGF, eNOS, HIF-1 alpha, and SIRT1, which can be completely eliminated by the inhibition of SIRT1 with 1 mmol/L nicotinamide. Conclusions The protection of rBMVECs from hypoxia/high-glucose induced impairment by DPP-4 inhibitors may be mediated by the SIRT1/HIF-1 alpha/VEGF pathway.