The structure of Langmuir monolayers and Langmuir–Blodgett (LB) films of a semi-fluorinated surfactant containing an intermediate hydrophilic group was investigated. The polar group in the middle of the molecule is shown to induce different conformational states depending on the substrate. At the air–water interface, the molecule adopts a folded shape in order to present its polar group to the water; this leads to a disordered phase. After transfer onto hydrophilic solid substrate (oxidized silicon wafers covered by a behenic acid bilayer), the molecule adopts a stretched conformation and hexagonal packing. Moreover, observed by atomic force microscopy, this LB film displays a labyrinthine morphology induced by the structure of the semi-fluorinated molecule. Finally, the influence of the conformational change of this molecule on its miscibility properties with behenic acid is studied.