〔Abstract〕 Objective To investigate the inhibitory effect of gingerol, an active component of ginger, on Staphylococcus aureus (S. aureus) infection, and to preliminarily explore its mechanism related to extracellular vesicles (EVs). Methods S. aureus infection models were established in HUVECs, Vero E6 cells, and C57BL/6 mice. Experimental groups included control, infection, and gingerol-treated groups. Bacterial load and VE-cadherin protein expression were detected using immunofluorescence and Western blot. EVs were isolated by size exclusion chromatography and characterized by transmission electron microscopy and nanoparticle tracking analysis. miRNA sequencing was performed on EVs. Results Gingerol treatment significantly reduced the bacterial load in both in vitro and in vivo infection models and upregulated VE-cadherin expression. miRNA sequencing of EVs revealed that S. aureus infection upregulated the expression of hsa-miR-320c, while gingerol treatment reversed this abnormal expression. Bioinformatic analysis further predicted that the target genes of hsa-miR-320c were significantly enriched in cell junction-related pathways. Conclusion Gingerol exhibits clear antibacterial and host-protective effects, by regulating hsa-miR-320c in EVs to maintain endothelial barrier integrity.