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-1alpha, signifying a critical role of the HIF pathway in cancer development. The recently identified third member of the human HIF-alpha family, HIF-3alpha, produces multiple splice variants that contain extra DNA binding elements and protein-protein interaction motifs not found in HIF-1alpha or HIF-2alpha. Here we report the molecular cloning of the alternatively spliced human HIF-3alpha variant HIF-3alpha4 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-3alpha4 suppresses the transcriptional activity of HIF-1 and siRNA-mediated knockdown of the endogenous HIF-3alpha4 increases transcription by hypoxia-inducible genes. HIF-3alpha4 itself is oxygen-regulated, suggesting a novel feedback mechanism of controlling HIF-1 activity. Furthermore, the expression of HIF-3alpha4 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-3alpha4 in vivo and underscore its potential significance in renal epithelial oncogenesis.