In this study, 25-hydroxycholesterol (25-OH), a biamphiphilic compound with a wide range of biological activities, has been investigated at the air/water interface. We were interested in how two hydroxyl groups attached at distal positions of the 25-OH molecule (namely, at C(3) in the sterane system and at C(25) in the side chain) influence its surface behavior. Apart from traditional Langmuir monolayers, other complementary surface-sensitive techniques, such as electric surface potential measurements, Brewster angle microscopy (BAM, enabling texture visualization and film thickness measurements), and polarization modulation-infrared reflection-absorption spectroscopy (PM-IRRAS), were applied. Experimental data have been interpreted with the aid of theoretical study. Our results show that 25-OH molecules in the monomolecular layer are anchored to the water surface alternatively with C(3) or C(25) hydroxyl groups. Theoretical calculations revealed that the populations of these alternative orientations were not equal and molecules anchored with C(3) hydroxyl groups were found to be in excess. As a consequence of such an arrangement, surface films of 25-OH are of lower stability as compared to cholesterol (considered as a non-oxidized analogue of 25-OH). Moreover, it was found that, upon compression, the transition from mono- to bilayer occurred. The molecular mechanism and interactions stabilizing bilayer structure were proposed. The explanation of the observed unusual surface behavior of 25-OH may contribute to an understanding of differences in biological activity between chain- and ring-oxidized sterols.