BackgroundSepsis is one of the leading causes of death worldwide and characterized by blood stream infections associated with a dysregulated host response and endothelial cell (EC) dysfunction. Ribonuclease 1 (RNase1) acts as a protective factor of vascular homeostasis and is known to be repressed by massive and persistent inflammation, associated to the development of vascular pathologies. Bacterial extracellular vesicles (bEVs) are released upon infection and may interact with ECs to mediate EC barrier dysfunction. Here, we investigated the impact of bEVs of sepsis-related pathogens on human EC RNase1 regulation.MethodsbEVs from sepsis-associated bacteria were isolated via ultrafiltration and size exclusion chromatography and used for stimulation of human lung microvascular ECs combined with and without signaling pathway inhibitor treatments.ResultsbEVs fromEscherichia coli,Klebsiella pneumoniaeandSalmonella entericaserovar Typhimurium significantly reduced RNase1 mRNA and protein expression and activated ECs, while TLR2-inducing bEVs fromStreptococcus pneumoniaedid not. These effects were mediated via LPS-dependent TLR4 signaling cascades as they could be blocked by Polymyxin B. Additionally, LPS-freeClearColi™ had no impact on RNase1. Further characterization of TLR4 downstream pathways involving NF-кB and p38, as well as JAK1/STAT1 signaling, revealed that RNase1 mRNA regulation is mediated via a p38-dependent mechanism.ConclusionBlood stream bEVs from gram-negative, sepsis-associated bacteria reduce the vascular protective factor RNase1, opening new avenues for therapeutical intervention of EC dysfunction via promotion of RNase1 integrity.Video AbstractSupplementary InformationThe online version contains supplementary material available at 10.1186/s12964-023-01131-2.