Abstract: Hypoxia is a general characteristic of most solid malignancies and intimately related to cancer progression.Homeostatic response to hypoxia is primarily mediated by hypoxia inducible factor-1α (HIF-1α) that elicits transcriptional activity through recruitment P300 coactivator.Targeting the interaction of HIF-α and P300 would thus constitute a novel approach for cancer treatment by suppressing tumor angiogenesis and metastasis.Here, a screening assay was developed for inhibitors targeting the interaction between HIF-1α and P300.The nucleotide sequence of human HIF-1α and P300 were cloned into pBIND and pACT vectors, named pBIND-HIF1α and pACT-P300.The interaction of HIF-1α and P300 was identified in HEK293 cell using mammalian two-hybrid system.And compound chetomin decreased their interaction in this mammalian two-hybrid system.We further verified HIF-1 inhibition effect of chetomin in U251-HRE cells.Therefore,we established a screening assay combined HIF-1α and P300 mammalian two-hybrid system and U251-HRE reporter assay for HIF-1 selective inhibitors.