Abstract Body (Do not enter title and authors here): Microvascular endothelial dysfunction is a strong predictor of future adverse cardiac events. We have previously shown that exposure to chronic estrogen induces microvascular endothelial dysfunction in isolated microvessels from otherwise healthy adults, with more extensive dysfunction exhibited in vessels from males. Further, we have demonstrated sex-specific, estrogen-induced increases in endothelial ceramide, a sphingolipid that when accumulated promotes microvascular endothelial dysfunction. The source of ceramide in response to estrogen remains unknown. Here we hypothesized that exogenous estrogen activates the ceramide-forming enzyme neutral sphingomyelinase (NSmase) in a sex-dependent manner. Sex-specific human umbilical vein endothelial cells (HUVECs) were cultured to passage 3 and treated with 17β-estradiol (E2, 100nM) or vehicle for 0, 1, and 2 minutes prior to collection and lysis of cells. NSmase activity was measured by the Neutral Sphingomyelinase Activity Assay Kit (Echelon Biosciences) according to manufacturer instructions. NSmase activity was greater in female compared to male HUVECs after 1 minute of treatment with E2 (normalized %change from control±SE; Untreated Control vs. 1 min E2; female:0.00±9.4% vs. 42.33±9.4%, n=6 vs. n=3, respectively, p=0.0174; male:-0.17±4.45 vs. 27.33±6.36, n=6 vs. n=3, respectively, p=0.1507; one-way ANOVA). Interestingly, NSmase activity was also significantly increased following 2 minutes of E2 treatment, an effect not observed in cells from females (normalized %change from control±SE; Untreated Control vs. 2 min E2; female:0.00±9.4% vs. 19.0±4.7%, respectively, n=6, p=0.26; male:-0.17±4.4 vs. 38.3±6.9, respectively, n=6, p=0.004). These data suggest that estrogen activates NSmase in a sex-dependent manner with an earlier, transient activation observed in females and a more sustained response in males. Collectively, this may further explain the sexually dimorphic response to estrogen and provide a potential therapeutic avenue to prevent microvascular damage in individuals chronically exposed to estrogen.