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Background. Staphylococcus aureus infection is one of the leading causes of morbidity in hospitalized patients in the United States. The secreted agent hemolysin alpha toxin (Hla) requires the receptor A Disintegrin And Metalloproteinase domain-containing protein 10 (ADAM10) to mediate its toxic effects; ADAM10 in turn activates the Notch pathway. Notch proteins function in developmental and pathological angiogenesis via the modulation of key pathways in endothelial (EC) and perivascular cells. Thus, we hypothesized that Hla would activate Notch in EC in vitro and in vivo. Methods. Human umbilical vein endothelial cells (HUVEC) were treated with recombinant Hla (rHla), Hla-H35L (genetically inactivated Hla), 5mM EDTA (a known Notch activator), or HBSS, and probed with a Luciferase reporter regulated by a Notch promoter. Mice engineered to express yellow fluorescent protein (YFP) upon Notch activation received a non-lethal daily subcutaneous injection of rHla (0.025 µg/5µL) or vehicle (PBS) and their retinal endothelial YFP interrogated at p6. 6 week old male Notch reporter mice received a 1-4 × 107 50 µl subcutaneous inoculation of S. aureus strains USA300/LAC (WT), or its isogenic Δhla mutant lacking Hla, and their skin biopsies were analyzed by histology after 36 hours, 8 and 16 days. Notch activation in endothelial cells of human liver sections from patients whose blood cultures were positive or negative for S. aureus was evaluated by immunohistochemistry. Results. Luciferase assays demonstrated that Hla (0.01 µg/mL) increased Notch activation by 1.75±0.5-fold as compared to HBSS controls (p ±3.1x106 vs. 7.7x10-5 ±6x105 YFP area/ DAPI area, p=0.002). Increased EC Notch activation in response to Hla was maintained 8 and 16 days after inoculation. IHC showed EC in human liver had higher Notch expression than controls. In sum, our results demonstrate that the S. aureus toxin Hla can potently activate Notch in endothelial cells, an effect which could have effects in development and pathological angiogenesis such as cancer. Citation Format: Sonia L. Hernandez, Mildred Nelson, Georgia Sampedro, Bianca Lec, Jared Emolo, Naina Bagrodia, Ann M. Defnet, Lydia Wu, Juliane Bubeck-Wardenburg, Jessica Kandel. Staphylococcus aureus alpha toxin activates Notch in endothelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2055.