Streptomyces actuosus ATCC 25421 was famous for producing thiopeptide nosiheptide, which has widely been used as a feed additive for the promotion of animal growth. Herein, we report the complete genome sequence of S. actuosus ATCC 25421, which consists of an 8,145,579-bp circular chromosome with a G+C content of 72.53?% containing 7?536 protein-coding genes. The antiSMASH 3.0 program was used to identify 49 biosynthetic gene clusters for putative secondary metabolites, including a putative lantipeptide gene cluster that showed 85?% similarity to the reported informatipeptin biosynthetic gene cluster, indicating that the putative lantipeptide gene cluster has the ability to generate the informatipeptin analogue. Compared with avermipeptin, the lantipeptide precursor peptide (termed avermipeptin B) from S. actuosus ATCC 25421 contains a 14-aa leader peptide and a 24-aa core peptide, in which Ile15 was different from Val15 in avermipeptin. We also deduced the structure and the biosynthetic mechanism of avermipeptin B. Heterologous expression of the avermipeptin B biosynthetic gene cluster in S. lividans TK24 was characterized by high-resolution mass spectrometry (ESI-MS/MS). Finally, we found that avermipeptin B displayed strong activity against Gram-positive strains. The genome sequence reported here can encourage us to mine novel secondary metabolites and investigate their biosynthetic mechanism in the future.