Objective Establish immortalized brain microvascular endothelial cells from tree shrews, clarify the effect of D-galactose on senescence of brain microvascular endothelial cells, and explore the potential mechanisms. Methods BMECs were transfected with a lentivirus carrying the SV40T gene, and immortalized cells were characterized by morphological observation, growth curve determination, immunofluorescence, and karyotype after expansion for > 50 passages. Next, BMECs were treated with various concentrations of D-gal, and the optimal concentration was determined by cell proliferation assays. Senescence was analyzed by staining with senescence-associated β-galactosidase. Expression of senescence-associated p53 was measured by Western Blot. Intracellular oxidative stress levels were measured by superoxide dismutase and lipid oxidation assays. Results Immortalized cells grew with consistent morphology throughout passages. Single cells were shaped as short spindles or polygonal with a typical paving stone shape. The cell growth curve showed cells were in the logarithmic growth phase on days 2 ~ 5. Cell growth peaked at day 6 and then the cells grew slowly to a plateau. High levels of tree shrew vWF, CD31, and SV40T were detected by immunofluorescence. Karyotyping showed that the number of chromosomes in immortalized cells matched the number of chromosomes in tree shrew (Tupaia belangeri chinensises). D-galactose reduced BMEC proliferation in time- and dose-dependent manners. In the experimental group, i.e. BMECs treated by D-gal at a concentration of 10 g/ L for 48 h, positive β-galactosidase staining was significantly increased, p53 was significantly increased, intracellular SOD activity was reduced, and MDA content was increased. Conclusions A tree shrew immortalized brain microvascular endothelial cell line was established successfully. The optimal concentration to establish an immortalized BMEC senescence model was 10 g/ L D-gal. The mechanism by which D-gal promotes cellular senescence in vitro may be mediated through promotion of oxidative stress and inhibition of cell proliferation.