Objective To investigate the effect of betaine in oxygen-glucose deprivation injury of rat brain microvascular endothelial cells（BMECs）， and to clarify the regulatory effect of betaine on phosphoinositide 3-kinase（PI3K）/protein kinase B（AKT） pathway. Methods Five SD rats aged 7 d were selected and the rat BMSEs were obtained. The oxygen-glucose deprivation model of rat BMECs was prepared under hypoxic and hypoglycemic conditions； the experiment was divdided into model group， and low dose， medium dose， and high dose of betaine groups and positive control group， at the same time， blank control group （without modeling） was set up. The BMECs in blank control group and model group were treated with fresh medium， the BMECs in positive control group were given a final concentration of 10 μmol·L^-1 nimodipine， and the BMECs in low， medium and high doses of betaine groups were treated with betaine at final concentration of 0.5， 1.0 and 2.0 mmol·L^-1， respectively. The survival rates of BMECs in various groups were determined by CCK-8 method at 12， 24 and 48 h after culture； the activities of lactate dehydrogenase （LDH） and the levels of adenine ribonucleoside triphosphate （ATP） in the rat BMECs in various groups were determined using kits， and the levels of tumor necrosis factor-α （TNF-α）， interleukin（IL）-6， IL-1β， and IL-18 in supernatants of the BMECs in various groups were determined by enzyme-linked immunosorbent assay （ELISA）； the activities of superoxide dismutase （SOD） and levels of malondialdehyde （MDA） in the BMECs in various groups were determined by kits； the transendothelial resistance （TEER） values of rat BMSCs in various groups were determined by TEER analyzer， and the horseradish peroxidase （HRP） permeabilities of BMECs in various groups were determined by an insertion cell culture apparatus. TUNEL staining was used to determine the apoptotic rates of rat BMECs in vaisous groups， and Western blotting method was used to determine the ratios of phosphory lated PI3K（p-PI3K）/PI3K and phosphorylated AKT（p-AKT）/AKT in the rat BMECs in various groups. Results Compared with blank control group， the survival rate of BMECs， activity of SOD， and level of ATP， value of TEER， and ratios of p-PI3K/PI3K and p-AKT/AKT of the rat BMECs in model group were significantly decreased （P<0.05）， while the activity of LDH， the levels of TNF-α， IL-6， IL-1β， IL-18， and MDA， the apoptotic rate of the BMECs， and HRP permeability were significantly increased（P<0.05）. Compared with model group， the survival rates of the BMECs， activities of SOD， and levels of ATP， values of TEER， and ratios of p-PI3K /PI3K and p-AKT /AKT of the BMECs in low， medium， and high doses of betaine groups and positive control group were significantly increased（P<0.05）， while the activities of LDH， the levels of TNF-α， IL-6， IL-1β， IL-18， and MDA， the apoptotic rates of BMECs and HRP permeabilities were significantly decreased（P<0.05）. Conclusion Betaine can significantly repair the oxygen-glucose deprivation/reperfusion injury in the rat BMECs， inhibit the oxidative damage and apoptosis of BMECs， and improve the permeability of the cells； its mechanism may be related to the regulation of the PI3K/AKT pathway.