Background: Enzalutamide is a potent second-generation androgen receptor (AR) antagonist with activity in metastatic castrate-resistant prostate cancer (CRPC). While enzalutamide is initially effective, disease progression inevitably ensues with the emergence of resistance. Intratumoral hypoxia is also associated with CRPC progression and treatment resistance. Given that both AR and HIF-1α are key regulators of these processes, dual targeting of both signaling axis represents an attractive therapeutic approach. Methods: Crosstalk of the AR and HIF-1α signaling pathways were examined in prostate cancer cell lines (LNCaP, 22Rv1) with assays measuring the effect of androgen and hypoxia on AR-dependent and hypoxia-inducible gene transcription, protein expression, cell proliferation, and apoptosis. Cells were stimulated with dihydrotestosterone (DHT) or the hypoxia mimetic cobalt chloride. HIF-1α inhibition was achieved by siRNA silencing HIF-1α or via chetomin, a disruptor of HIF-1α-p300 interactions. Results: In prostate cancer cells, gene expressions of AR targets (KLK3, FKBP5, TMPRSS2) were repressed by HIF-signaling; conversely, HIF-1α target expressions (VEGF, ENO1, LDHA) were induced by DHT-mediated AR signaling. Treatment of CRPC cells with enzalutamide and chetomin or HIF-1α siRNA attenuated AR-regulated and HIF-1α-mediated gene transcription. The combination of enzalutamide and HIF-1α inhibition was more effective than either treatment alone. Similarly, the combination also reduced VEGF protein levels. HIF-1α siRNA synergistically enhanced the inhibitory effect of enzalutamide on cell growth in LNCaP and enzalutamide-resistant 22Rv1 cells. Additionally, HIF-1α siRNA synergistically increased enzalutamide-induced apoptosis. Conclusions: Combination of enzalutamide with HIF-1α inhibition resulted in synergistic inhibition of AR-dependent and gene specific HIF-dependent expression, prostate cancer cell growth, and apoptosis.