Hygromycin A, an antibiotic produced by Streptomyces hygroscopicus NRRL 2388, consists of three unusual moieties, namely 5-dehydro-a-L-fucofuranose, (E)-3-(3,4-dihydroxyphenyl)-2-methylacrylic acid, and an aminocyclitol (2L-2-amino-2-deoxy-4,5-O-methylene-neo-inositol). Recently we reported the hygromycin A biosynthetic gene cluster (hyg) and proposed its biosynthetic pathway. The structure-activity relationship studies have revealed the importance of structurally unique aminocyclitol moiety with methylene bridge for its antibacterial actitivities. In order to explore the biosynthetic pathway of this moiety, a series of gene deletion studies have been carried out, and provided both intermediates in the biosynthetic pathway and a new analog. In this study, we will report role of hyg6, hyg7, hyg8, hyg17, hyg18 and hyg25 in the biosynthesis of aminocyclitol moiety of hygromycin A.