A 45 kb DNA sequencing analysis from Streptomyces hygroscopicus 5008 involved in validamycin A (VAL-A) biosynthesis revealed 16 structural, 2 regulatory, and 5 genes related to transport, transposition/integration, tellurium resistance, and another 4 genes with no obvious identity. The VAL-A biosynthetic pathway was proposed, with assignment of the required genetic functions confined in the sequenced region. A cluster of eight reassembled genes was found to support the VAL-A synthesis in a heterologous host, S. lividans 1326. In vivo inactivation of the putative glycosyltransferase gene (valG) abolished the final attachment of glucose for VAL production, and resulted in the accumulation of the VAL-A precursor, validoxylamine, while the normal production of VAL-A could be restored by complementation with valG. The role of ValG in the glycosylation of validoxylamine to VAL-A was demonstrated in vitro by enzymatic assay.