[Objective] Blasticidin S (BS) is a hexose-containing peptidyl-nucleoside antibiotic widely used as fungicidal in agriculture and as an effective selection reagent in eukaryotic cells. Genetic manipulation in the native producer of BS was never successful, which hinder the studies of BS biosynthesis. We recently engrafted the BS biosynthetic gene cluster into the chromosome of Streptomyces lividans and achieved heterologous production of BS. In this study, we tried to localize the boundary of BS gene cluster to identify the essential genes, providing functional insights into its remaining unknown biosynthetic steps. [Methods] PCR-targeting was used to carry out gene replacements in S. lividans-derived BS producer. The mutants were fermented and assayed by LC-MS for their capability of producing BS. [Results] We defined the boundary of the essential BS biosynthetic gene cluster that contains ten genes blsD to blsM. Disruption of blsK leads to the accumulation of demethylblasticidin S (DBS) and BS. [Conclusion] Ten genes blsD to blsM are sufficient for BS biosynthesis. BlsK was responsible for adding leucine to the β-amino group of arginine-derived side chain of DBS to form leucyldemthylblasticidin S (LDBS), There are two biosynthetic pathway for BS: DBS is directly methylated to BS; the alternative is that DBS is first converted to LDBS that is then methylated and finally hydrolyzed into BS.