<jats:title>Summary</jats:title><jats:p>Antibiotic‐producing microorganisms have evolved several self‐resistance mechanisms to prevent auto‐toxicity. Overexpression of specific transporters to improve the efflux of toxic antibiotics has been found one of the most important and intrinsic resistance strategies used by many <jats:italic>Streptomyces</jats:italic> strains. In this work, two ATP‐binding cassette (ABC) transporter‐encoding genes located in the natamycin biosynthetic gene cluster, <jats:italic>scnA</jats:italic> and <jats:italic>scnB</jats:italic>, were identified as the primary exporter genes for natamycin efflux in <jats:italic>Streptomyces chattanoogensis</jats:italic> L10. Two other transporters located outside the cluster, a major facilitator superfamily transporter Mfs1 and an ABC transporter NepI/II were found to play a complementary role in natamycin efflux. ScnA/ScnB and Mfs1 also participate in exporting the immediate precursor of natamycin, 4,5‐de‐epoxynatamycin, which is more toxic to <jats:italic>S. chattanoogensis</jats:italic> L10 than natamycin. As the major complementary exporter for natamycin efflux, Mfs1 is up‐regulated in response to intracellular accumulation of natamycin and 4,5‐de‐epoxynatamycin, suggesting a key role in the stress response for self‐resistance. This article discusses a novel antibiotic‐related efflux and response system in <jats:italic>Streptomyces</jats:italic>, as well as a self‐resistance mechanism in antibiotic‐producing strains.