<p>Magnetite nanoparticles (MNPs, Fe₃O₄) have gained considerable interest for various biomedical applications. Gramicidin A (GrA) is a linear pentadecapeptide that plays an important role in modifying the biophysical and biological properties of membranes. Elucidating how GrA modulates the interaction between MNPs and biomembranes is essential for deepening our understanding of membrane biophysics and guiding the development of nanomedicine. We investigated the effects of GrA on anionic MNPs-induced deformation, membrane permeation, and the stability of cell-mimetic giant unilamellar vesicles (GUVs) under physiological conditions. The membranes of the GUVs were prepared using a combination of negatively charged lipid, neutral lipid, and varying GrA. The degree of deformation (e.g., compactness) decreased with increasing GrA. The leakage of encapsulated calcein from the GUVs indicated membrane poration caused by the adsorption of MNPs into the membranes. The leakage rate initially decreased and then increased as GrA rose. To assess membrane stability, we examined constant tension-induced rupture of GUVs by varying GrA using the micropipette technique. The rupture rate constant also showed a biphasic response, initially decreasing and then increasing with the increase of GrA at a constant membrane tension. Thus, GrA modulates the mechanical stability of membranes, along with MNPs-induced vesicle deformation and lipid membrane poration.</p>