As antibiotics are always toxic to the antibiotic-producing strains themselves, most Streptomyces strains have evolved several self-resistance mechanisms, among which the antibiotic efflux system is understood best and is commonly found. Among the efflux systems, the ATP-binding cassette (ABC) transporter superfamily and the major facilitator superfamily (MFS) are two important transporter families. In this work, the ABC transporters and the MFS transporters from the four reported natamycin-producing Streptomyces strains have been investigated in order to clarify whether these Streptomyces strains share similar efflux strategies for natamycin metabolism. Fifty-one groups of homologous exporter genes were identified as shared by four strains. Differential transcriptional analysis between the natamycin-producing strain Streptomyces chattanoogensis L10 and its ΔscnS0 mutant, which produces no natamycin, reveals that the expression levels of 25 of the above groups of genes were observably changed. The production of natamycin declined over 30% after solely knocking out several of these 25 groups of genes in S. chattanoogensis L10. This indicates that these transporters participate in the efflux of molecules related to natamycin biosynthesis. Our study is the first to demonstrate that the exporters participating in a particular antibiotic metabolism can be excavated and identified quickly by the strategy of genome mining and homologous comparison in the antibiotic-producing strains, leading to deeper understanding of the complex self-resistance mechanisms in Streptomycetes.