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Adhesion to the intestinal tract provides the foundation for Lactobacillus to exert its benefits. Vacuum freeze-drying (VFD) is currently one of the main processing methods for Lactobacillus products. Therefore, the effects of VFD on the adhesion and survival of <i>Lactiplantibacillus plantarum</i> 67 were investigated in this study. The results show that <i>L. plantarum</i> 67 exhibits remarkable tolerance following successive exposure to simulated saliva, gastric juice and intestinal juice, and also has a strong adhesion ability to Caco-2 cells. The adhesion and survival rates of <i>L. plantarum</i> 67 significantly decreased after VFD in phosphate-buffered saline (PBS), whereas they significantly increased in protective agents (PAs) (<i>p</i> < 0.05). Scanning electron microscope observations show that <i>L. plantarum</i> 67 aggregated more to Caco-2 cells in PAs than in PBS, and its shape and size were protected. Proteomics detection findings indicated that differentially expressed proteins (DEPs) related to adhesins and vitality and their pathways in <i>L. plantarum</i> 67 were significantly affected by VFD (<i>p</i> < 0.05). However, the expression of DEPs (such as cold shock protein, cell surface protein, adherence protein, chitin-binding domain and extracellular transglycosylase, membrane-bound protein) was improved by PAs. Compared with PBS, the PAs significantly adjusted the phosphotransferase system and amino sugar and nucleotide sugar metabolism pathways (<i>p</i> < 0.05). VFD decreased the adhesion and vitality of <i>L. plantarum</i> 67, while the PAs could exert protective effects by regulating proteins and pathways related to adhesion and vitality.