Excessive production of renal reactive oxygen species (ROS) play a major role in diabetic kidney disease (DKD). Here, we provide key novel findings demonstrating the predominant pathological role of the prooxidant enzyme NADPH oxidase-NOX5 in DKD, independent of the previously characterised NOX4 pathway. In diabetic patients, we found increased expression of renal NOX5 in association with enhanced ROS formation and upregulation of ROS-sensitive factors EGR-1 (early growth response 1), PKC-α (protein kinase C- α) and a key metabolic gene involved in redox balance, TXNIP (thioredoxin-interacting protein). In preclinical models of DKD, overexpression of <i>NOX5</i> in <i>Nox4</i> deficient mice enhances kidney damage by increasing albuminuria and augmenting renal fibrosis and inflammation via enhanced ROS formation and the modulation of EGR1, TXNIP, ERK1/2, PKC-α and PKC-ε. In addition, the only first in class NOX inhibitor, GKT137831 appears to be ineffective in the presence of NOX5 expression in diabetes. In vitro, silencing of NOX5 in human mesangial cells attenuated high glucose induced upregulation of EGR1, PKC-α, and TXNIP as well as markers of inflammation (TLR4 and MCP-1) and fibrosis (CTGF and collagens I and III) via reduction in ROS formation. Collectively, these findings identify NOX5 as a superior target in human DKD compared to other NOX isoforms such as NOX4 which may have been overinterpreted in previous rodent studies.