The transcriptional factor, hypoxia inducible factor-1 (HIF-1), is a promising target for cancer chemotherapy. From an actinomycete, verucopeptin (1) was identified as a HIFI signaling inhibitor. By a combination of chemical degradation and spectroscopic analyses, the absolute stereochemistry of metabolite 1 was determined to be 10R, 15S, 16S, 235, 27S, 28R, 31S, 33S, 35R. Moreover, metabolite 1 was revealed to attenuate the HIF-1 alpha and mTORC1 pathway, indicating that verucopeptin (1) would be a potent lead compound for anticancer chemotherapy.