von Willebrand factor (vWF) is a large multimeric sialoglycoprotein that plays key roles in normal haemostasis, inflammation regulation, angiogenesis and cancer metastasis in mammals. The gene, protein sequences and functions of vWF in flounder Paralichthys olivaceus (PovWF) were analysed in this study. PovWF possesses an 8550-bp open reading frame (ORF) that encodes a 2849 amino acid protein. PovWF mRNA is highly expressed in the heart and gill, followed by the intestine, skin, spleen, kidney, muscle and liver. PovWF positive cells are mainly endothelial cells (ECs), predominantly located along the inner lining of blood vessels, enclosing the bloodstream. After being infected with hirame novirhabdovirus (HIRRV), flounder exhibits a dark body colour, congested fins and visceral membranes. Histopathologic analysis revealed that the ECs of diseased fish had compromised integrity, accompanied by a significant increase in number of cells within blood vessels. Immunofluorescence and ultrastructural studies showed that virions infect ECs can induce morphological and functional alterations, which lead to the release of vWF and facilitate the migration of neutrophils into tissues to exert antiviral functions. This research pinpoints the role of vWF in the immune response to HIRRV infection in teleost. It offers an in-depth and all-encompassing understanding of the pathophysiological interaction between HIRRV and endothelial cells during invasive infections in fish.