Nuclear pore complexes (NPCs) are supra-molecular protein assemblies which mediate nucleocytoplasmic transport highly selectively. The NPC barrier thereby restricts the efficiency of gene therapy by preventing exogenously applied therapeutic macromolecules from nuclear delivery. A controlled breakdown of the NPC barrier may lead to an increased efficiency of gene therapy. Amphipathic alcohols, such trans-cyclohexane-1,2-diol (TCHD), are known to break down the NPC barrier and yet the underlying mechanism remains unclear. A controlled breakdown of the NPC barrier, especially while avoiding cytotoxic adverse effects at the same time, requires a precise understanding of the structural organization of the NPC barrier. The present study sheds light onto the enigmatic structural organization of the NPC permeability barrier. Furthermore, we apply TCHD and surprisingly show that a release of barrier forming proteins from the NPC, rather than a disruption of their interaction with each other, is critical to break down the NPC barrier. NPC proteins interestingly do not contribute equally to the formation of the barrier. A controlled sequential release of different barrier-forming proteins from the NPC through progressively increasing TCHD concentrations results in a gradual breakdown of the NPC permeability barrier ultimately leading to its complete collapse. This implies that a controlled release of proteins that contribute most to the formation and maintenance of the NPC barrier can facilitate access of therapeutic macromolecules into the nucleus while minimizing the cytotoxicity at the same time.