The most prevalent mutations associated with the development of clear-cell renal cell carcinoma (CC-RCC) are the loss-of-function mutations of von Hippel-Lindau (VHL) tumor suppressor gene. These mutations invariably result in an inappropriate accumulation of HIF-alpha due to a failure of VHL as a substrate-recognition component of an E3 ubiquitin ligase complex to target HIFalpha for oxygen-dependent ubiquitin-mediated destruction. Stabilization of HIF-2alpha, but not HIF-1alpha, is the critical oncogenic event upon the functional loss of VHL in the development of CC-RCC. Here, we show that HIF-3alpha4, an alternatively spliced variant of human HIF-3alpha with similar domain structure as the murine inhibitory PAS protein (IPAS), forms an abortive transcriptional complex with HIF-2alpha and prevents the engagement of HIF-2 to the hypoxia-responsive elements (HREs) located in the promoter/ enhancer regions of hypoxia-inducible genes. In addition, the re-expression of HIF-3alpha4 in VHL-null 786-O CC-RCC cells via adenovirus decreases the endogenous expression of HIF-2-driven gene expression and suppresses the growth of 786-O tumor xenografts in SCID mice. These results suggest that HIF-3alpha4 is a naturally occurring dominant-negative HIF-3alpha splice isoform with tumor suppressive activity and support the targeted delivery of HIF-3alpha4 as a potential therapeutic option to curtail HIF-dependent tumor progression.