Hexanal is a phytochemical with antimicrobial activity.However,its antibacterial effect and mechanism against Vibrioparahaemolyticus (V. parahaemolyticus) remain unclear. The study aims to elucidate the associated mechanismusing tandem mass tag quantitative proteomics and non-targeted metabolomics.Hexanal treatment reduced intracellular ATP concentration, increasedmembrane permeability, and destroyed the morphology and ultrastructureof V. parahaemolyticus cells. Proteomicsand metabolomics data indicated that 572 differentially expressedproteins (DEPs) and 241 differential metabolites (DMs) were identifiedin hexanal-treated V. parahaemolyticus. These DEPs and DMs were involved in multiple biological pathwaysincluding amino acid metabolism, purine and pyrimidine biosynthesis,etc. Bioinformatics analysis revealed that hexanal damaged the structureand function of cell membranes, inhibited nucleotide metabolism, anddisturbed carbohydrate metabolism and tricarboxylic acid cycle (TCA)cycle, which ultimately resulted in growth inhibition and bacterialdeath. The study is conducive to better understand the mode of actionof hexanal against V. parahaemolyticus and offers experimental foundation for the application of hexanalas the antibacterial agent in the seafood-associated industry.