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Lactic acid bacteria (LAB) are used in fermented foods to overcome the shortcomings of traditional fermentation methods, such as long cycles and poor safety. However, the application of LAB in low-temperature foods is limited by their growth temperature. This study aimed to quantitatively compare the proteins of the YT2 strain acclimated at 37, 28, 20, 15, 8, and 4 °C (YT-4) and that grown at 37 °C (YT-37) using tandem mass tag (TMT) proteomics and bioinformatics analysis to elucidate the potential mechanism of cryotolerance of P. pentosaceus YT2. Altogether, 1416 quantitative proteins were identified, of which 747 were differentially abundant between the YT-4 and YT-37 strains. Functional enrichment analysis showed that the upregulated proteins were mainly involved in nucleotide transport, metabolism, translation, ribosomal structure and biogenesis, transcription, replication, recombination, and repair, while the downregulated proteins were involved in lipid, amino, and carbohydrate transport and metabolism. These results were validated for eight upregulated and five downregulated proteins using parallel reaction monitoring (PRM). Our study analyzed the potential mechanism underlying the improved cryotolerance of P. pentosaceus YT2 at 4 °C, which may enable the utilization of LAB in low-temperature applications. • The effect of cold acclimation on P. pentosaceus YT2 was revealed. • A total of 747 differentially abundant proteins were identified. • The expression changes of 15 differentially abundant proteins were validated. • Cold acclimation can improve the cryotolerance of YT2. [ABSTRACT FROM AUTHOR]