Abstract Body: Acute ischemic stroke (AIS) increases plasma ceramides that correlate with the prevalence and severity of neurological and motor deficits in patients. We sought to determine cause and effect. Adult male C57Bl6 mice challenged with 60-min transient middle cerebral artery (MCA) occlusion (tMCAO) followed by 23-h reperfusion (R) displayed increased (p<0.05) ceramides (LC-MS/MS) in plasma and endothelial cells (ECs) isolated from ipsilesional hemispheres vs. age/sex matched sham-operated mice. Further, compared to vehicle treatment, endogenous ceramide accrual blunted: MCA and parenchymal arteriolar vasodilation ex vivo (isobaric myography); and mouse brain microvascular EC mitochondrial efficiency (Seahorse XF Bioanalyzer; both p<0.05). These findings motivated the hypothesis that cerebral EC ceramide generation worsens AIS outcomes. Serine palmitoyl transferase (SPT) catalyzes the first step in de novo ceramide biosynthesis. AAV-BR1-Cre or AAV-BR1-Null virus was deployed to adult male mice engineered to allow for overexpression of a fusion construct comprising the 3 SPT subunits, enabling induction of cerebral EC ceramides (fSPT mice). Twenty-one days later, Sptlc1 and Sptlc2 mRNA were elevated (p<0.05) in the cerebral EC-enriched fraction of AAV-BR1-Cre (Cre-fSPT mice) vs. AAV-BR1-Null (Null-fSPT) mice, confirming efficacy and specificity of delivery. In additional mice, 60-min tMCAO reduced ipsilesional perfusion (laser speckle contrast imaging) similarly between groups, but reperfusion at 23-h was lower, and tissue injury (TTC staining) and neurobehavioral (Bederson test, mNSS) and motor (grip test, grip strength) deficits were more severe (all p<0.05), in Cre-fSPT vs. Null-fSPT animals. Degs1 is the gene encoding dihydroceramide desaturase 1 (DES1), the fourth step in ceramide biosynthesis. Delivering AAV-BR1-Cre (Cre-Degs1 mice) but not AAV-BR1-Null (Null-Degs1 mice) virus to Degs1 floxed mice reduced (p<0.05) Degs1 mRNA expression in the cerebral EC -enriched fraction and thereby ceramide accrual. In additional mice, AIS reduced perfusion similarly between groups, but ipsilesional reperfusion at 23-h was higher, and tissue injury, neurobehavioral and motor deficits were less severe (p<0.05) in Cre-Degs1 vs. Null-Degs1 mice. In conclusion, these results indicate that AIS-induced cerebral EC ceramide generation is sufficient and necessary to worsen outcomes and might represent an innovative therapeutic target to combat AIS complications.