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The aim of this research was to investigate the antimicrobial characteristics and mechanism of hesperetin against Alicyclobacillus acidoterrestris vegetative cells. The results presented show that hesperetin had effective antimicrobial activity on Alicyclobacillus acidoterrestris vegetative cells, minimum inhibition concentration (MIC) of 0.0625 g/L, and minimum bacterial concentration (MBC) greater than 2 g/L. Moreover, treatment of hesperetin caused significant damage to cell integrity, preventing the growth of Alicyclobacillus acidoterrestris vegetative cells, enhancing the leakage of nucleic acid and proteins, and destroying the vegetative cell morphology. To further investigate the mechanism, transcriptomic analysis was carried out, and 3056 differentially expressed genes (DEGs) were detected. Gene ontology (GO) enrichment analysis revealed that hesperetin inhibits Alicyclobacillus acidoterrestris by affecting the intracellular nitrogen metabolism and amino acid metabolism. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis explained that hesperetin was also able to prevent the growth of Alicyclobacillus acidoterrestris by affecting the processes of nutrient transport, energy metabolism, and flagella motility. These results provide new insights into the antimicrobial effects and mechanism of hesperetin against Alicyclobacillus acidoterrestris, which provides a new method for inactive Alicyclobacillus acidoterrestris in the juice industry.