Over-activation of NADPH oxidase and uncoupling of nNOS could be important mechanisms causing oxidative stress and increased oxygen consumption commonly associated with reduced oxygen availability in the diabetic kidney. Therefore, we investigated the role of each of these mechanisms for the increased oxygen consumption by isolated proximal tubular cells (PTC) from diabetic rats. Oxygen consumption was measured in freshly isolated PTC from streptozotocin-induced diabetic rats with and without chronic treatment with the NADPH oxidase inhibitor apocynin, or the specific nNOS inhibitor S-methyl-L-thiocitrulline (SMTC), or in combination and compared to controls (n=7–10 per group). PTC from untreated diabetic rats had increased oxygen consumption compared to controls (40.6±7.9 vs. 10.9±2.0 nmol/mg protein/min). All treatments reduced the diabetes-induced increase in oxygen consumption (apocynin 10.5±1.7, SMTC 19.7±3.0 and apocynin+SMTC 21.6±3.6 nmol/mg protein/min). Neither of apocynin nor SMTC had any effect on the oxygen consumption in cells pre-incubated with ouabain, an inhibitor of active electrolyte transport. Oxidative stress, estimated from thiobarbituric acid reactive substances (TBARS) and protein carbonylation, was elevated in the diabetic kidney and inhibited by the applied treatments. The increased oxygen consumption by the diabetic PTC correlated with increased protein expressions of p47^phox and nNOS, and the treatments prevented these increases. This study shows that increased NADPH oxidase activity and uncoupling of nNOS result in increased oxygen consumption due to elevated oxidative stress in the diabetic kidney. The effects of apocynin and SMTC are not additive, suggesting a unifying pathway, which warrants further studies.