The substantial availability of hypoxia-inducible factor 1 (HIF-1) for pathophysiological states, such as malignancies and ischemia, is primarily regulated post-translationally through the ubiquitin proteolytic system. The balance between degradation and stabilization of HIF-1α protein is determined by specific E3 ligases. In our search for new E3 ligases that might affect HIF-1α protein expression, we studied the effects of beta-transducin repeat-containing protein (β-TrCP) on the hypoxic pathway in cancer cells. β-TrCP is overexpressed in many tumors and regulates various cellular processes through mediating the degradation of important targets. Unexpectedly, we found that β-TrCP overexpression increases HIF-1α protein expression level as well as HIF-1 transcriptional activity by stabilizing HIF-1α protein and preventing its ubiquitination and proteasomal degradation in prostate cancer cells. By using a proteomic approach, we succeeded in demonstrating that β-TrCP interferes with the association between HIF-1α and HSP70/CHIP, a HIF-1α established E3 ligase complex. Whereas the E3 ligase activity of β-TrCP is well known, antagonizing another E3 ligase is a new mechanism of action of this important E3. We suggest that destroying or suppressing β-TrCP and thereby interrupting the HIF-1 pathway, could be valuable antitumor therapy.