The structure of an ornithine (Orn)-free Gramicidin S (GS) analogue, cyclo(Val–Nle–Leu–D-Phe–Pro)₂ (NGS), was studied. Its circular dichroism (CD) spectrum showed that NGS has a structure similar to GS, though the value of [θ] indicated smaller β-turn and sheet populations. This is probably because the Nle side chain could not form intramolecular hydrogen bonds stabilizing the sheet structure. The chemical shift perturbation of αH and J_NH–αH were similar in GS and NGS. Three independent NGS molecules formed intramolecular β-sheet structures in crystal. The turn structures of D-Phe-Pro moieties were classed as type II′ β-turns, but one part was unclassed. The molecules were arranged in a twisting manner, which resulted in the formation of a helical sheet. Similar structural characteristics were observed previously in a Leu-type, Orn-free GS analogue and in GS trifluoroacetic acid salt.