NW-G01 is a novel cyclic hexapeptide antibiotic produced by Streptomyces alboflavus 313. Its relative structure was established by HR-ESI-MS, IR, 1D and 2D NMR techniques, the absolute structure was determined using a combination of single-crystal X-ray diffraction and Marfey's method finally. The antibiotic consists of L-valine, (3S)- and (3R)-piperazic acids, N-methyl-D-alanine and (2S,3aR,8aS)-6-chloro-3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole-2-carboxylic acid.