Hypoxia-inducible transcription factors (HIFs) regulate hundreds of genes involved in cellular adaptation to reduced oxygen availability. HIFs consist of an O2-labile α-subunit (primarily HIF-1α and HIF-2α) and a constitutive HIF-1β subunit. In normoxia the HIF-α subunit is hydroxylated by members of a family of prolyl-4-hydroxylase domain (PHD) proteins, PHD1-3, resulting in recognition by von Hippel-Lindau protein, ubiquitination and proteasomal degradation. In contrast, reduced oxygen availability inhibits PHD activity resulting in HIF-1α stabilisation and nuclear accumulation. Nuclear import of HIF-1α mainly depends on classical nuclear localisation signals (NLS) and involves importin α/β heterodimers. Recently, a specific inhibitor of nuclear import has been identified that inhibits importin α/β-dependent import with no effects on a range of other nuclear transport pathways involving members of the importin protein family. In this study we evaluated the physiological activity of this importin α/β-inhibitor (Ivermectin) in the hypoxia response pathway. Treatment with Ivermectin decreases binding activity of HIF-1α to the importin α/β-heterodimer. Moreover, HIF-1α nuclear localisation, nuclear HIF-1α protein levels, HIF-target gene expression, as well as HIF-transcriptional activity are reduced upon Ivermectin treatment. For the first time, we demonstrate the effect of specific importin α/β-inhibition on the hypoxic response on the molecular level.