The protein kinase C (PKC)-β isoform has been implicated to play a pivotal role in the development of diabetic kidney disease. We tested this hypothesis by inducing diabetic nephropathy in PKC-β–deficient (PKC-β^−/−) mice. We studied nondiabetic and streptozotocin-induced diabetic PKC-β^−/− mice compared with appropriate 129/SV wild-type mice. After 8 weeks of diabetes, the high-glucose–induced renal and glomerular hypertrophy, as well as the increased expression of extracellular matrix proteins such as collagen and fibronectin, was reduced in PKC-β^−/− mice. Furthermore, the high-glucose–induced expression of the profibrotic cytokine transforming growth factor (TGF)-β1 and connective tissue growth factor were significantly diminished in the diabetic PKC-β^−/− mice compared with diabetic wild-type mice, suggesting a role of the PKC-β isoform in the regulation of renal hypertrophy. Notably, increased urinary albumin-to-creatinine ratio persisted in the diabetic PKC-β^−/− mice. The loss of the basement membrane proteoglycan perlecan and the podocyte protein nephrin in the diabetic state was not prevented in the PKC-β^−/− mice as previously demonstrated in the nonalbuminuric diabetic PKC-α^−/− mice. In summary, the differential effects of PKC-β deficiency on diabetes-induced renal hypertrophy and albuminuria suggest that PKC-β contributes to high-glucose–induced TGF-β1 expression and renal fibrosis, whereas perlecan, as well as nephrin, expression and albuminuria is regulated by other signaling pathways.