Abstract <p><i>Alicyclobacillus acidoterrestris</i> causes pasteurized acidic juices spoilage, resulting in a significant decline in juice quality and causing economic losses. Exploration of <i>A. acidoterrestris</i> in response to acid stress could help control contamination caused by the bacteria. In this study, the mechanism of <i>A. acidoterrestris</i> in response to acid stress was studied by quantitative phosphoproteomics technique. Results showed that the phosphorylation of 40 proteins in <i>A. acidoterrestris</i> was closely related to the regulation of acid stress. The KEGG pathway enrichment analysis showed that the quorum sensing pathway, which might be involved in the perception of <i>A. acidoterrestris</i>, was mainly enriched. We found that the upregulation of Spo0A and YidC phosphorylationmay resist acid stress by forming spores. The phosphorylation level of pyruvate kinase increased, which may improve bacterial acid stress resistance through the formation of energy supply. The phosphorylation level of ABC transporter permease was significantly upregulated, which may be part of the cell adaptation adjustment and contribute to the survival of <i>A. acidoterrestris</i> under acid stress. In summary, the molecular mechanism of acid stress regulation of <i>A. acidoterrestris</i> was proposed via quantitative phosphoproteomics, which provided a theoretical and experimental basis for further investigation of the acid resistance mechanism of <i>A. acidoterrestris</i>.</p> Key points <p>• <i>Spo0 A, Yidc proteins may be the key regulatory proteins for acid stress response.</i></p> <p>• <i>ABC transporters are beneficial to the survival of the bacteria under acidic stress.</i></p> <p>• <i>A. acidoterrestris may sense and transmit pH signal through quorum sensing pathway.</i></p>