Corneal endothelial decompensation (CED) is the major cause of the long-term graft failure, but the underlying mechanisms remain unclear. The purpose of this study was to characterize the proteomic profile in CED aqueous humor (AH) after penetrating keratoplasty (PKP). We collected AH samples (n = 6/group) from CED patients underwent PKP and cataract patients, respectively. The label-free quantitative proteomic analysis was performed to identify the differentially-expressed proteins (DEPs). The biological functions of DEPs were evaluated using Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genome (KEGG) analysis. The protein-protein interaction (PPI) network construction was employed to distinguish the hub proteins of DEPs, and the selected proteins were validated by parallel reaction monitoring (PRM). The human peripheral blood mononuclear cells (PBMCs) were adopted to investigate the effect of biglycan (BGN) on inflammatory response, and the subsequent outcomes of inflammation on human corneal endothelial cells (HCECs). A total of 174 DEPs were identified in CED AH of patients underwent PKP, including 102 up-regulated proteins and 72 down-regulated proteins. Bioinformatics analysis revealed the significant enrichment of cytokine-mediated signaling pathway and extracellular matrix (ECM) organization in the up-regulated proteins, as well as the alterations of cellular components, including the increase of collagen and complement component C1 complex, and reduction in extracellular exosomes. A hub protein cluster of 15 proteins was determined by Molecular Complex Detection (MCODE), including FN1, BGN, COMP, COL11A1, COLA3A1, and COL1A1. Moreover, BGN promoted pro-inflammatory cytokine (such as TNF-α, IL-1β and IL-6) production in PBMCs through NF-κB signaling pathway, which subsequently resulted in HCECs death. These findings provided a systemic protein profile of AH in CED patients after corneal transplantation, with the alterations implicated in cytokine-mediated signaling, ECM, complement system, and exsomes. The identified proteins and signaling pathways probably paved the novel insight into understanding the pathogenesis of the disease. •The protein profile of aqueous humor in CED was quite different from cataract group.•The up-regulated proteins are functionally associated with inflammatory response.•Atypical enrichment of ECM and C1 complex in the up-regulated proteins of CED.•The extrinsic component of (plasma) membrane, and exosomes were remarkably reduced.