Activation of protein kinase C (PKC) isoforms has been implicated in the pathogenesis of diabetic nephropathy. We showed earlier that PKC-α is activated in the kidneys of hyperglycemic animals. We now used PKC-α^−/− mice to test the hypothesis that this PKC isoform mediates streptozotocin-induced diabetic nephropathy. We observed that renal and glomerular hypertrophy was similar in diabetic wild-type and PKC-α^−/− mice. However, the development of albuminuria was almost absent in the diabetic PKC-α^−/− mice. The hyperglycemia-induced downregulation of the negatively charged basement membrane heparan sulfate proteoglycan perlecan was completely prevented in the PKC-α^−/− mice, compared with controls. We then asked whether transforming growth factor-β1 (TGF-β₁) and/or vascular endothelial growth factor (VEGF) is implicated in the PKC-α–mediated changes in the basement membrane. The hyperglycemia-induced expression of VEGF165 and its receptor VEGF receptor II (flk-1) was ameliorated in PKC-α^−/− mice, whereas expression of TGF-β₁ was not affected by the lack of PKC-α. Our findings indicate that two important features of diabetic nephropathy—glomerular hypertrophy and albuminuria—are differentially regulated. The glucose-induced albuminuria seems to be mediated by PKC-α via downregulation of proteoglycans in the basement membrane and regulation of VEGF expression. Therefore, PKC-α is a possible therapeutic target for the prevention of diabetic albuminuria.