Glomerular integrity and functions are maintained by growth factor signaling. Heparan sulfate, the major component of glomerular extracellular matrixes, modulates growth factor signaling, but its roles in glomerular homeostasis are unknown. We investigated the roles of heparan sulfate 6- O-endosulfatases, sulfatase (Sulf)1 and Sulf2, in glomerular homeostasis. Both Sulf1 and Sulf2 were expressed in the glomeruli of wild-type (WT) mice. Sulf1 and Sulf2 double-knockout (DKO) mice showed glomerular hypercellularity, matrix accumulation, mesangiolysis, and glomerular basement membrane irregularity. Platelet-derived growth factor (PDGF)-B and PDGF receptor-β were upregulated in Sulf1 and Sulf2 DKO mice compared with WT mice. Glomeruli from Sulf1 and Sulf2 DKO mice in vitro stimulated by either PDGF-B, VEGF, or transforming growth factor-β similarly showed reduction of phospho-Akt, phospho-Erk1/2, and phospho-Smad2/3, respectively. Since glomerular lesions in Sulf1 and Sulf2 DKO mice were reminiscent of diabetic nephropathy, we examined the effects of Sulf1 and Sulf2 gene disruption in streptozotocin-induced diabetes. Diabetic WT mice showed an upregulation of glomerular Sulf1 and Sulf2 mRNA by in situ hybridization. Diabetic DKO mice showed significant increases in albuminuria and serum creatinine and an acceleration of glomerular pathology without glomerular hypertrophy; those were associated with a reduction of glomerular phospho-Akt. In conclusion, Sulf1 and Sulf2 play indispensable roles to maintain glomerular integrity and protective roles in diabetic nephropathy, probably by growth factor modulation.