In this study, we provide the first experimental evidence that colloidal hydrophilic magnetite nanoparticles can penetrate through bilayer lipid membrane in a non-uniform stationary magnetic field. Hydrophilic ligand-free cationic colloidal magnetite nanoparticles with an average diameter of 4 nm were added to the surrounding aqueous solution on one side of the azolectin membrane. An external non-uniform magnetic field ensured the attraction of superparamagnetic magnetite nanoparticles to the membrane, resulting in the formation of a near-membrane charged layer of cationic nanoparticles resulting in the initial polarization of the membrane. As a result of the passage of magnetite nanoparticles through the membrane, the polarization of the membrane decreases, and the membrane becomes depolarized. Independent methods were used to detect magnetite nanoparticles that passed through the lipid membrane including transmission electron microscopy and energy-dispersive x-ray spectroscopy. The discovered effect may be due to the following factors and interactions of nanoparticles. Interaction of magnetic nanoparticles with external inhomogeneous magnetic field provides localization of nanoparticles on the membrane surface. Collective interactions between nanoparticles, as well as their interactions with external electric and magnetic fields, lead to the formation of magnetite nanoparticle aggregates. Interaction of nanoparticles with the membrane lipid matrix leads to the formation of organic–inorganic complexes in which the polar surface of nanoparticles is enveloped by a lipid layer. The penetration of nanoparticles through the membrane is caused by the interaction of organic–inorganic complexes of nanoparticles and their aggregates with local intramembrane and near-membrane electric and magnetic fields.