The voltage-dependent anion channel (VDAC) resides in the outer mitochondrial membrane and can adopt a closed or open configuration, most likely depending on whether the N-terminal segment (NTS) occupies the pore or protrudes into the cytoplasm. In this study, we calculate the free energy of releasing the NTS from the pore using molecular dynamics simulation. This is complicated by the flexible nature of the NTS, in particular its disordered structure in aqueous solution compared to the pore lumen. We carried out potential of mean force calculations using enhanced sampling or conformational restraints to address the conformational sampling problem. For the binding to the VDAC pore, two systems were considered, featuring either the native VDAC system or a modified system where the NTS is detached from the pore, that is, noncovalently bound in the pore lumen. The calculated free energies required to translocate the NTS from the pore into the solvent moiety are 83.8 or 74.3 kJ mol−1, respectively. The dissociation pathway in VDAC presents two in-pore minima, separated by a low free energy barrier and a membrane-bound intermediate state. Since we observe small changes in pore shape along the NTS dissociation pathway, we suggest that rigidification of the VDAC pore might impair NTS dissociation. The stability of the membrane-bound state of the VDAC NTS is confirmed by independent molecular dynamics simulations showing spontaneous membrane binding of a NTS-derived peptide as well as nuclear magnetic resonance experiments where chemical shift perturbations of the NTS-derived peptide evidence binding to phospholipid nanodiscs.