Objective To screen the differential proteins related to postharvest cap opening and reveal the molecular mechanism of postharvest opening of Agaricus bisporus. Methods Fruiting bodies before storage (0 d) and immediately after velum broken (6 d) were used as the experimental materials to screen differentially expressed proteins related to the postharvest velum broken and cap opening of Agaricus bisporus by iTRAQ proteomics followed by further bioinformatics analysis and identification including GO (Gene Ontology) analysis and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis. Results A total of 808 differentially expressed proteins, including 285 up-regulated proteins and 523 down-regulated proteins, were identified between two mushroom groups stored on day 0 and day 6. Comparison with the genome database revealed that only 38 of the screened differential proteins were annotated, and the annotated proteins were mainly closely related to biological processes such as arginine metabolism, nucleic acid metabolism, glucose metabolism and oxidative reactions. The enrichment of GO function and KEGG pathway showed that the differential proteins related to cap opening were mainly enriched in the plasma membrane, mitochondria, and ribosomal cell structure, and were involved in the pathways of cyanoamino acid metabolism, phenylpropane biosynthesis, glucose metabolism, and arginine and proline metabolism, which indicated that these differential proteins and metabolic pathways were related to the cap opening of postharvest Agaricus bisporus mushrooms. Conclusion The signaling pathways and metabolic processes involved in differential proteins, especially arginine metabolism, play an important role in the postharvest cap opening process of Agaricus bisporus.