This work was designed to investigate differential expression proteins at different mature stages of Volvariella volvacea using isobaric tags for relative and absolute quantification (iTRAQ)-coupled two-dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS) proteomics approach. The proteins extracted from V. volvacea at different mature stages were analyzed by SDS-PAGE. After that, the tandem mass spectrometry data obtained from 2D LC-MS/MS were used to search using MASCOT search engine. The functional analysis of differential expression proteins was conducted using KEGG. Results showed that 1 039 protein groups, included a total of 2 335 unique peptides, were identified. Among them, 1 030 protein groups were provided quantitative information. Compared with egg stage, 85 up-regulated and 103 down-regulated significantly differential proteins were found at elongation and mature stages of V. volvacea. Analysis of KEGG pathways showed that 68 different expression proteins at different development stages of V. volvacea were involved in 45 unique metabolism pathways from four model fungi of Agaricales, Coprinopsis cinerea, Laccaria bicolor, Moniliophthora perniciosa and Schizophyllum commune. This work displayed the whole picture of metabolic networks mapped by different expression proteins at different mature stages of V. volvacea. Our results revealed that iTRAQ-coupled 2D LC-MS/MS is a powerful method for isolating and identifying protein groups of V. volvacea at different development stages.