We have shown previously that septin 9 isoform 1 (SEPT9_i1) protein associates with hypoxia-inducible factor (HIF)-1alpha to augment HIF-1 transcriptional activity by driving its importin-alpha-mediated nuclear translocation. Using in vitro and in vivo binding assays we identified that HIF-1alpha interacts with importin-alpha5 and importin-alpha7 in prostate cancer cells but only importin-alpha7 interacts with SEPT9_i1. The interaction with importin-alpha7 was dependent on the first 25 amino acids of SEPT9_i1 that are unique compared to other members of the mammalian septin family. Depletion of endogenous importin-alpha7 reduced HIF-1alpha levels in the nucleus. Our results provide evidence that there are importin-alpha specificities in the cytosolic/nuclear translocation process of HIF-1alpha protein, which may act differently under certain pathophysiological circumstances where SEPT9_i1 is overexpressed.