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Vesicle-associated membrane protein (VAMP)-associated proteins (VAP27s), which are widely expressed in plants and animals, play an important role in metabolism, physiology, growth, and development, disease resistance, and immunity. While the function of this family has been elucidated in model plants like Arabidopsis thaliana and tomato, its role in grapevine remains unclear. In this present study, 12 vesicle-associated protein-membrane protein genes were identified in the grapevine genome by bioinformatics, designated as the VAP27 gene family. A phylogenetic tree, encompassing 53 genes from three model plants, Arabidopsis thaliana, Oryza sativa, and Solanum lycopersicum, revealed the subdivision of the VAP27 gene family into three subfamilies, each presumably serving different functions, besides localizing in endoplasmic reticulum, individual members also localize in nucleus. Additionally, we compared the transcriptional levels and subcellular localizations of the VvVAP27 family members across different plant tissues (flower, leaf, seed, root, fruit, tendril, and stem), indicating site-specific functionalities for different gene members. To investigate the responsiveness of the VAP27 gene family to pathogen infection, particularly Plasmopara viticola on host plants, we analyzed the expression patterns of VAP27 genes post-infection. Our findings revealed divergent expression profiles among different members at different stages of infection. The gene family responded to the infection of downy mildew on grapevine and could inhibit the spread of Phytophthora capsici lesions in Nicotiana benthamiana. These results provide an important basis for further studies delving into the functions of the VAP27 gene family in plant growth and disease resistance.