Nox4 is a hydrogen peroxide-producing NADPH oxidase highly expressed in the kidney which has been linked to epithelial cell injury and diabetic-induced cellular dysfunction in cultured cells. The role of the enzyme for renal pathology in vivo, however, is unclear. To address this, three experimental animal models of renal injury (streptozotocin diabetes I, unilateral ureteral ligation (UUO), and 5/6 nephrectomy (5/6Nx)) were studied in either Nox4-inducible (Nox4(*/*)) or constitutive knockout (Nox4(-/-)) mice. Nox4 contributed more than 80% of diphenylene iodonium-sensitive H(2)O(2) formation of freshly isolated tubules determined by Amplex Red assay. In streptozotocin diabetes, acute deletion of Nox4 by tamoxifen-activated cre-recombinase increased albuminuria, whereas matrix deposition was similar between WT and Nox4(*/*) mice. Interestingly, renal Nox4 expression, mainly localized to tubular cells, decreased in the course of diabetes and this was not associated with a compensatory upregulation of Nox1 or Nox2. In the UUO model, renal expression of ICAM1, connective tissue growth factor, and fibronectin were higher in kidneys of Nox4(*/*) than control mice. Also in this model, levels of Nox4 decreased in the course of the disease. In the 5/6Nx model, which was performed in SV129 and SV129-Nox4(-/-) mice, no difference in the development of hypertension and albuminuria was found between the strains. Collectively, the first in vivo data of the kidney do not support the view that Nox4 is a main driver of renal disease. It rather appears that under specific conditions Nox4 may even slightly limit injury and disease progression.