Based on genome mining, a new antibacterial peptide named actinokineosin was isolated from a rare actinomycete Actinokineospora spheciospongiae. The amino acid sequence of the C‐terminus of actinokineosin was established by TOF‐MS/MS experiments. The amino acid sequence in the macrolactam ring was determined by TOF‐MS/MS analyses after cleavage with BNPS‐skatole and successive trypsin treatment. As a result of an antibacterial assay using a paper disk, actinokineosin showed antibacterial activity against Micrococcus luteus at a dosage of 50 μg per disk. From the genome sequence data of A. spheciospongiae, the biosynthetic gene cluster of actinokineosin was found and was indicated to consist of 10 genes. Among the genes, the gene aknA encoded the precursor of actinokineosin and the genes including aknC, aknB1 and aknB2 were proposed as modification enzymes to give mature actinokineosin. Significance and Impact of the Study Genome mining is a powerful tool to find new bioactive compounds from the genome database. In this report, we succeeded in isolation and structure determination of a new antibacterial peptide named actinokineosin based on genome mining. Significance and Impact of the Study: Genome mining is a powerful tool to find new bioactive compounds from the genome database. In this report, we succeeded in isolation and structure determination of a new antibacterial peptide named actinokineosin based on genome mining.