A universal response to changes in cellular oxygen tension is governed by a family of heterodimeric transcription factors called hypoxia‐inducible factor (HIF). Tumor hypoxia, as well as various cancer‐causing mutations, has been shown to elevate the level of HIF‐1α, signifying a critical role of the HIF pathway in cancer development. The recently identified third member of the human HIF‐α family, HIF‐3α, produces multiple splice variants that contain extra DNA binding elements and protein‐protein interaction motifs not found in HIF‐1α or HIF‐2α. Here we report the molecular cloning of the alternatively spliced human HIF‐3α variant HIF‐3α4 and show that it attenuates the ability of HIF‐1 to bind hypoxia‐responsive elements located within the enhancer/promoter of HIF target genes. The overexpression of HIF‐3α4 suppresses the transcriptional activity of HIF‐1 and siRNAmediated knockdown of the endogenous HIF‐3α4 increases transcription by hypoxia‐inducible genes. HIF‐3α4 itself is oxygen‐regulated, suggesting a novel feedback mechanism of controlling HIF‐1 activity. Furthermore, the expression of HIF‐3α4 is dramatically down‐regulated in the majority of primary renal carcinomas. These results demonstrate an important dominant‐negative regulation of HIF‐1‐mediated gene transcription by HIF‐3α4 in vivo and underscore its potential significance in renal epithelial oncogenesis. Maynard, M. A., Evans, A. J., Hosomi, T., Hara, S., Jewett, M. A. S., Ohh, M. Human HIF‐3α4 is a dominant‐negative regulator of HIF‐1 and is down‐regulated in renal cell carcinoma. FASEB J. 19, 1396–1406 (2005)