The hypoxia inducible factor (HIF) transcriptional complex is central to hypoxia signaling in cells and activates the expression of target genes involved in angiogenesis, oxygen transport, iron metabolism, glycolysis, glucose uptake, growth factor signaling, apoptosis, invasion and metastasis. Deregulation of the HIF pathway occurs in many human cancers and has been shown to drive both angiogenesis and tumor progression and furthermore it correlates with the severity of tumor grade. Inhibition of HIF activity has been shown to significantly inhibit tumor growth in vivo. HIF is therefore an attractive therapeutic target. The HIF-1 complex consists of α and β subunits. Both HIF-1α and HIF-1β (also known as aryl hydrocarbon nuclear receptor translocator, ARNT) are members of the basic helix-loop-helix Per/Arnt/Sim (bHLH-PAS) transcription factor family. Activation of HIF-1 is dependent on the availability of the HIF-1α subunit, while HIF-1β is usually constitutively expressed in cells. HIF-1α protein is tightly regulated in normoxia via the oxygen-dependent degradation domain (ODD). The ODD domain contains a number of prolyl residues that are recognised and hydroxylated by specific prolyl hydroxylase domain (PHD) enzymes. This results in binding of the von Hippel-Lindau protein (pVHL), an E3 ligase which targets the HIF-1α protein for degradation via the proteasome pathway. HIF-1α protein is rapidly induced in response to hypoxia and growth factor stimulation by an increase in stability and synthesis respectively. Upon induction, HIF-1α translocates to the nucleus where it binds to HIF-1β to form the HIF-1 complex. Transactivation of HIF-1 target genes is dependent upon binding to the hypoxia responsive element (HRE) found within their promoter region. The HIF-1 complex recruits a number of co-activators, such as p300/CBP thus transactivating the expression of a multitude of target genes, the most prominent being vascular endothelial growth factor (VEGF). In human cancer, changes in microenvironmental stimuli, genetic instability and mutations leading to loss of tumor suppressor function or oncogenic activation can lead to the overexpression of HIF-1α. Indeed, it is now clear that deregulation of the HIF pathway occurs in response to many of the key genetic abnormalities that lead to cancer. There are several sites in the HIF pathway that are potential intervention points for inhibition by small molecule inhibitors. These include inhibition of HIFα stability or protein synthesis or interference of HIF-dependent interactions. A number of small-molecule inhibitors of HIF have been described, although their exact mechanism of action remains to be understood. In addition, cell-based high-throughput screens are also being used to identify novel small molecule inhibitors of HIF. These systems generally utilize cells transfected with multiple HREs linked to a specific reporter gene construct. This allows efficient screening of large libraries of compounds for HIF-inhibitory activity. We have recently developed and validated a cell-based assay which we used to perform a high-throughput screen (HTS) to identify novel small molecule inhibitors of the HIF pathway. Extensive hit evaluation has allowed us to identify a potent inhibitor of tumor cell growth in vitro and in vivo. In addition our deconvolution analysis has identified a mechanism of action for one of our novel compounds. These studies will be presented in detail in the context of our current understanding of HIF cancer biology.