Background <p>Androgen deprivation and androgen receptor (AR) antagonists are essential treatments for prostate cancers in the clinic. However, after an initially effective response, most tumors become resistant to androgen deprivation therapies. Resistance often arises from AR mutations, resulting in ligand-independent activation and nuclear translocation of AR.</p> Methods <p>This study investigated the effects of nuclear import inhibition on AR signaling and enzalutamide resistance. We utilized nuclear import inhibitors (importazole and ivermectin) and KPNB1 (importin β) knockdown in prostate cancer cell models. We assessed AR subcellular localization, AR-dependent transactivation (transcriptional activity), cell viability and apoptosis in cells expressing wild-type AR, the AR-F876L mutant, or the constitutively active AR splice variant AR-V7.</p> Results <p>Inhibition of nuclear import suppressed AR-dependent transactivation, increased cytoplasmic AR, and enhanced the action of enzalutamide by promoting the apoptosis of prostate cancer cells. While enzalutamide became an AR agonist in cells harboring the mutated AR (F876L), inhibition of nuclear import inhibited enzalutamide-induced nuclear localization and transactivation of the mutated AR and reduced the viability of AR-F876L-expressing cells. Both importazole and ivermectin attenuated the transcriptional activation induced by a constitutively active AR splice variant (AR-V7), while enzalutamide showed no effect.</p> Conclusion <p>Inhibition of nuclear import can overcome enzalutamide resistance and may serve as a novel strategy for prostate cancer treatment.</p>