Inactivation of the von Hippel-Lindau tumor suppressor (VHL) is an early event in >60% of sporadic clear cell renal cell carcinoma (RCC). Loss of VHL E3 ubiquitin ligase function results in accumulation of the α-subunit of the hypoxia-inducible heterodimeric transcription factor (HIF-α) and transcription of an array of genes including vascular endothelial growth factor, transforming growth factor-α, and erythropoietin. Studies have shown that HIF-α can be alternatively activated by reactive oxygen species. Nox4 is an NADP(H) oxidase that generates signaling levels of superoxide and is found in greatest abundance in the distal renal tubules. To determine if Nox4 contributes to HIF activity in RCC, we examined the impact of Nox4 expression on HIF-α expression and transactivation. We report here that small inhibitory RNA (siRNA) knockdown of Nox4 in 786-0 human renal tumor cells expressing empty vector (PRC) or wild-type VHL (WT) results in 50% decrease in intracellular reactive oxygen species as measured by a fluorescent 2′,7′-dichlorofluorescin diacetate assay, and >85% reduction in HIF2-α mRNA and protein levels by quantitative reverse transcription-PCR and Western blot analysis. Furthermore, expression of the HIF target genes, vascular endothelial growth factor, transforming growth factor-α, and Glut-1 was abrogated by 93%, 74%, and 99%, respectively, after stable transfection with Nox4 siRNA relative to nontargeting siRNA, as determined by quantitative reverse transcription-PCR. Thus, renal Nox4 expression is essential for full HIF2-α expression and activity in 786-0 renal tumor cells, even in the absence of functional VHL. We propose the use of Nox4 as a target in the treatment of clear cell RCC.