Background: The activation of mineralocorticoid receptor (MR) is known to be involved in kidney disease progression. MR antagonists (MRAs) have been reported to have beneficial effects on various kidney diseases. However, it is still unclear which mechanism underlies kidney and glomeruli injury related to MR activation. Recently, we demonstrated that aldosterone promotes proteoglycan synthesis in mitral valve interstitial cells and its association with the development of cardiac valvular disorder. Proteoglycans are glycosylated molecules sequestered in the extracellular matrix (ECM), interacting with other components of ECM. In addition, some proteoglycans are known to interact with several bioactive partners to activate cell signaling. Since increased expression of certain proteoglycans has been reported in several kidney diseases, we investigated the association between the proteoglycans expression and kidney injury upon aldosterone/MR pathway activation. Methods: We analyzed the alteration and localization of proteoglycan expression in the kidney of uninephrectomy/aldosterone/salt (NAS)-treated mice. The MRA eplerenone was administered to these mice to evaluate the contribution of the MR pathway in NAS-induced kidney injury. Glomeruli samples were isolated from the kidneys to assess the localized expression of proteoglycans and the glomerular injury. Additionally, we performed cellular experiments using isolated peritoneal macrophages to assess the direct effect of a proteoglycan biglycan on macrophages. Results: NAS treatment enhanced renal fibrosis and urinary albumin levels. Renal expression of proteoglycans was increased. Among various proteoglycans, biglycan showed increased expression, especially in the glomeruli. Isolated glomeruli samples revealed glomerular macrophage infiltration, accompanying enhanced expression of TNF-α (tumor necrosis factor-alpha), iNOS (inducible nitric oxide synthase), Nox2 (NADPH oxidase 2), CCL3 (C-C motif chemokine ligand 3), and phosphorylated NF-κB (nuclear factor-kappa B). The expression of CD86, a surface marker for M1 macrophage, also increased by 11.5-fold in the glomeruli of NAS-treated mice. These changes were blunted by eplerenone. Treatment with purified biglycan induced expression of TNF-α, iNOS, Nox2, and CCL3 in the isolated macrophages. An inhibitor of toll-like receptor 4 (TLR4) or NF-κB prevented this increase, indicating that the effect of biglycan stimulation is dependent on the TLR4/NF-κB pathway. Conclusion: This study identified the proteoglycan biglycan and its downstream TLR4/NF-κB/CCL3 cascade as a novel mediator of MR activation-induced glomerular injury.