Introduction: Agents which potently inhibit transforming growth factor-β (TGFβ) have limited clinical use due to unacceptable side effects. One pathway by which latent TGFβ1 is converted to its active form is through binding to the cationic-independent mannose 6-phosphate receptor (CI-M6PR). We have previously shown that the CI-M6PR inhibitor PXS25, has anti-fibrotic properties in human kidney tubular (HK-2) cells under high glucose conditions, but its clinical use is limited by low bioavailability. Aim: To determine the antifibrotic effects of PXS64, a pro-drug of PXS-25, in in vivo and in vitro models of renal fibrosis. Method: A 7 day unilateral ureteric obstruction (UUO) model was examined in mice randomized to the following groups: (i) Sham operated control ; (ii) UUO; (iii) UUO + PSX64 (10mg/kg) and (iv) UUO + Telmisartan (3mg/kg). Kidney tissue was analysed morphometrically and mRNA (real time PCR) and protein (Western blot and immunohistochemistry) for fibrotic and inflammatory markers were determined. HK-2 cells were exposed to TGFβ1 (1ng/ml) +/- PXS64 (10 μmol/L) for 24 hours. Results: The mRNA expression of ColIV and FN in kidney tissue was lower in animals treated with PXS64 (p<0.05 vs sham). The mRNA expression of TGFβ1 and monocyte chemoattractant protein-1 (MCP1) were effectively suppressed by PSX64 and telmisartan (both p<0.05 vs sham). In addition, mice treated with PXS64 had lower FN and ColIV protein expression determined by immunohistochemistry. PXS-64 inhibited TGFβ1-induced mRNA and protein expression of ColIV and FN in HK-2 cells (P < 0.05 vs control) but it did not inhibit TGFβ1-induced phosphorylated Smad2/3, AKT or PAK protein levels. Conclusion: PSX64 is an effective agent in preventing kidney fibrosis in the UUO model. PXS64 attenuates TGFβ1-induced pro-fibrotic and pro-inflammatory markers in the proximal tubular cells independent of Smad2/3, AKT and PAK signaling pathways.