<jats:p>The solution structure of filipin III, an antifungal polyene macrolide biosynthesized by <jats:italic>Streptomyces filipinensis</jats:italic> and widely used for the detection and the quantitation of cholesterol in biomembranes, has been calculated with a set of geometrical restraints derived from <jats:sup>1</jats:sup>H NMR in DMSO‐<jats:italic>d</jats:italic> <jats:sub>6</jats:sub> at 25°C. Filipin III appears as a rod‐shaped molecule of 18 Å length. Its amphiphilic structure is made of an all‐<jats:italic>syn</jats:italic> 1,3‐polyol motif, stabilized by intramolecular hydrogen bonds on one side, and a conjugated pentaene moiety on the other side of the molecule. The overall shape is comparable to cholesterol, and the molecular structure of filipin III affords a first molecular basis to the comprehensive understanding of the interactions possible in the filipin III–cholesterol complex which is still unknown at the atomic resolution.