Cholesterol seals human cellular membranes against water and water-soluble solutes by filling the voids between phospholipid molecules. Also, its presence modulates function of membranes as semipermeable barriers. It is known that even small modifications in the chemical structure of cholesterol can lead to strong alteration of membrane properties. In this work, we apply fluorescence methods as well as molecular dynamics simulations to characterize changes in lipid membrane permeability induced by cholesterol oxidation. The studied 7β-hydroxycholesterol (7β-OH-chol) and 27-hydroxycholesterol (27-OH-chol) represent two distinct groups of oxysterols, namely, ring- and tail-oxidized cholesterols, respectively. Our previous research showed that oxidation of cholesterol tail only marginally affects the structure of lipid bilayer. On the other hand, it disturbs membrane dynamics introducing new rapid type of trans-bilayer movement of the sterol – bobbing. Herein we show that bobbing of 27-OH-chol accelerates fluorescence quenching of NBD-lipid in the inner leaflet of liposomes by dithionite added to the liposomal suspension. Systematic experiments using fluorescence quenching spectroscopy and microscopy led to conclusion that the presence of 27-OH-chol increases membrane permeability to dithionite anion. Atom-scale molecular dynamics simulations demonstrate that this oxysterol also facilitates water transport across the membrane. All these findings support the view that oxysterol bobbing introduces sequential disturbance in the hydrophobic core of the membrane and is capable of aiding water molecules, as well as small water-soluble molecules, across lipid bilayer. This permeabilization can have many important consequences for eukaryotic organisms. The effects described for 27-OH-chol were not observed for 7β-OH-chol – the ring-oxidized sterol.