Abstract Body: Cerebral small vessel disease (SVD) contributes to approximately 50% of human dementias world-wide and pose a burden to the health care system worldwide. Early endothelial/blood-brain barrier (BBB) dysfunction is an important contributor to the pathogenesis of SVD. Sphingosine-1-phosphate (S1P), a membrane-derived signaling sphingolipid, regulates the BBB integrity by binding to its receptor isoform 1 (S1PR1) on endothelial cells. We previously showed that activation of S1PR1 using SEW2871, a selective S1PR1 agonist, reversed the loss of cerebral capillary endothelial S1PR1 and BBB integrity in a rat model of SVD. The current study evaluated multi-doses of SEW2871 as a novel therapy by targeting BBB damage for SVD. Male and female spontaneously hypertensive rat stroke-prone (SHRSPs) were subjected to unilateral carotid artery occlusion (UCAO) followed by Japanese permissive diet (JPD) for 35 days. Three doses of SEW2871 (0.5, 1.0 and 5.0 mg/kg) were tested. Neuropathological outcomes were assessed with MRI, immunohistochemistry, and Morris water maze (MWM). Body weight, oxygen saturation, and heart rate were evaluated weekly. SEW2871 plasma concentrations were measured at 2, 5, and 24 hours after injection. MRI showed increased BBB permeability, white and gray matter lesions (WML and GML) and decreased cerebral blood flow at 2 and 5 weeks after UCAO/JPD onset. T2 and fractional anisotropy images showed that three doses of SEW2871 demonstrated protective effect on WML and GML with most significant improvements in 1.0 mg/kg group. Dynamic contrast-enhanced MRI and arterial spin labeling (ASL) maps showed improved BBB leakage and CBF most remarkable in SEW2871 0.5 and 1.0 mg/kg groups compared to the vehicle rats. Furthermore, MWM test demonstrated that treatment with SEW2871 1.0 mg/kg presented significant improvement in spatial memory compared to UCAO/JPD group. Post-mortem immunohistochemical staining for IgG and Iba-1 showed BBB leakage and active microglia in lesioned WM and GM, which were significantly attenuated by SEW2871 1.0 mg/kg compared to vehicle rats, supporting the results obtained from MRI. Our data suggested that long-term treatment with SEW2871 reduced BBB permeability leading to improvement of brain lesions, CBF reduction, and cognitive impairment. The results also indicated that the effectiveness of SEW2871 in all doses tested, while 1.0 mg/kg showed a most remarkable protection against the neuropathological cascades associated with SVD.