Hyperglycemia and hypoxia have independent and convergent roles in the development of renal disease. Transforming growth factor-β₁ (TGF-β₁) is a key cytokine promoting the production of extracellular matrix proteins. The cationic-independent mannose 6-phosphate receptor (CI-M6PR) is a membrane protein that binds M6P-containing proteins. A key role is to activate latent TGF-β₁. PXS25, a novel CI-MPR inhibitor, has antifibrotic properties in skin fibroblasts, but its role in renal fibrosis is unclear. The aim was to study the role of PXS25 in matrix protein production under high glucose ± hypoxic conditions in human proximal tubule (HK-2) cells. HK-2 cells were exposed to high glucose (30 mM) ± 100 μM PXS25 in both normoxic (20% O₂) and hypoxic (1% O₂) conditions for 72 h. Cellular fibronectin, collagen IV, and matrix metalloproteinase-2 (MMP-2) and MMP-9 were assessed. Total and active TGF-β₁ were measured by ELISA. High glucose and hypoxia independently induced TGF-β₁ production. Active TGF-β₁, but not total TGF-β₁ was reduced with concurrent PXS25 in the presence of high glucose, but not in hyperglycemia+hypoxia conditions. Hyperglycemia induced fibronectin and collagen IV production (P < 0.05), as did hypoxia, but only hyperglycemia-induced increases in matrix proteins were suppressed by concurrent PXS25 exposure. High glucose induced MMP-2 and -9 in normoxic and hypoxic conditions, which was not modified in the presence of PXS25. High glucose and hypoxia can independently induce endogenous active TGF-β₁ production in human proximal tubular cells. PXS25 inhibits conversion of high glucose-induced release of active TGF-β₁, only in the absence of hypoxia.