Sphingosine‐1‐phosphate receptor (S1PR) modulators elicit neuroprotection during stroke, however the mechanism(s) associated with beneficial outcome remains unknown. Endothelial dysfunction is a key pathophysiological contributor in acute ischemic stroke. Here, we researched the vascular specific role of the S1PR1 ligand, ozanimod, on endothelial function and health following hypoxia plus glucose deprivation (HGD), an in vitro ischemic‐like model. Specifically, we investigated whether ozanimod (0.5 nM) alters human male brain microvascular endothelial cell (HBMEC) proliferation, morphology, and integrity. HBMECs were exposed to HGD (1% O2) or normoxia (21% O2) for 3h at passage 7 using a humidified chamber (BioSpherix®) housed within a 5% CO2 incubator. For the proliferation studies, cells were treated with bromodeoxyuridine and incorporation of the synthetic nucleoside analog during normoxic or HGD exposure was measured via flow cytometric analysis. Morphology was assessed post normoxic or HGD exposure in HBMECs stained with crystal violet and visualized with brightfield microscopy. Trans endothelial electrical resistance was measured following HGD or normoxia exposure using polyethylene terephthalate 8 well plates and electric cell‐substrate impedance sensing (ECIS® Z Theta). Additionally, we investigated the impact of HGD on S1PR1 and cyclooxygenase‐2 levels using standard immunoblotting techniques. In some experiments, selective blockade with the antagonist, W146, verified S1PR1 activation involvement. We observed that S1PR1 was basally expressed in HBMECs and HGD increased expression suggesting S1PR1 as a potential target during acute ischemic injury. Ozanimod attenuated HGD‐mediated decreases in HBMEC proliferation and W146 reversed this response. S1PR1 blockade had no effect on cell proliferation following normoxia. Ozanimod also preserved HGD‐induced morphological changes. During electric cell‐substrate impedance assessment, ozanimod attenuated HGD mediated reduction in endothelial cell resistance and W146 abolished this effect suggesting S1PR1 dependency. Concomitantly, we observed that HGD reduced expression levels of zonula occludens (ZO)‐1 and claudin‐5. Ozanimod also decreased HGD‐mediated increases in proinflammatory mediator expression of cyclooxygenase‐2. In conclusion, ozanimod elicited protection against HGD in HBMECs and may play an important beneficial role against acute ischemic damage by improving brain endothelial integrity and viability via selective S1PR1 activation.Support or Funding InformationUniversity of Arizona Valley Research Partnership Grant VRP37 P2 (RJG, FS), American Heart Association 19AIREA34480018 (RJG).