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Background: <i>Pleurotus ostreatus</i> is a popular edible mushroom in East Asian markets. Research on the responses of <i>P. ostreatus</i> under different carbon dioxide concentrations is limited. Methods: Label-free LC-MS/MS quantitative proteomics analysis technique was adopted to obtain the protein expression profiles of <i>P. ostreatus</i> fruiting body pileus collected under different carbon dioxide concentrations. The Pearson correlation coefficient analysis and principal component analysis were performed to reveal the correlation among samples. The differentially expressed proteins (DEPs) were organized. Gene ontology analysis was performed to divide the DEPs into different metabolic processes and pathways. Results: The expansion of stipes was inhibited in the high CO₂ group compared with that in the low CO₂ group. There were 415 DEPs (131 up- and 284 down-regulated) in <i>P. ostreatus</i> PH11 treated with 1% CO₂ concentration compared with <i>P. ostreatus</i> under atmospheric conditions. Proteins related to hydrolase activity, including several amidohydrolases and cell wall synthesis proteins, were highly expressed under high CO₂ concentration. Most of the kinases and elongation factors were significantly down-regulated under high CO₂ concentration. The results suggest that the metabolic regulation and development processes were inhibited under high CO₂ concentrations. In addition, the sexual differentiation process protein Isp4 was inhibited under high CO₂ concentrations, indicating that the sexual reproductive process was also inhibited under high CO₂ concentrations, which is inconsistent with the small fruiting body pileus under high CO₂ concentrations. Conclusions: This research reports the proteome analysis of commercially relevant edible fungi <i>P. ostreatus</i> under different carbon dioxide concentrations. This study deepens our understanding of the mechanism for CO₂-induced morphological change in the <i>P. ostreatus</i> fruiting body, which will facilitate the artificial cultivation of edible mushrooms.