Surfactin is an efficient biosurfactant excreted by different strains of Bacillus subtilis. Our study provides a molecular view of the temperature dependence of the structure and the interfacial properties of un-ionized surfactin micelles. The overall size and shape, the surface area, the radial density distribution of the micelles, the conformation of the hydrocarbon chain, and the intramolecular/intermolecular hydrogen bonds formed in surfactin molecules were investigated. The micelles were mostly in sphere shapes, and the radii of surfactin micelle were estimated to be around 2.2 nm. The peptide rings occupied most of the surface of the micelles. Small amounts of β-turn and γ-turn structures were found in the conformations of the peptide rings. When the temperature increased, the shape of the peptide rings became planar; the solvent accessible surface area decreased as temperature dehydration occurred. At 343 K some hydrocarbon chains reversed their orientation (flip-flopped). In addition, the stability of the hydrogen bond interactions in the micelles decreases with the increasing temperature.