Streptomyces albidoflavusJ1074 is a popular platform to discover novel natural products via the expression of heterologous biosynthetic gene clusters (BGCs). There is keen interest in improving the ability of this platform to overexpress BGCs and, consequently, enable the purification of specialized metabolites. Mutations within generpoBfor the β-subunit of RNA polymerase are known to increase rifampicin resistance and augment the metabolic capabilities of streptomycetes. Yet, the effects ofrpoBmutations on J1074 remained unstudied, and we decided to address this issue. A target collection of strains that we studied carried spontaneousrpoBmutations introduced in the background of the other drug resistance mutations. The antibiotic resistance spectra, growth, and specialized metabolism of the resulting mutants were interrogated using a set of microbiological and analytical approaches. We isolated 14 differentrpoBmutants showing various degrees of rifampicin resistance; one of them (S433W) was isolated for the first time in actinomycetes. TherpoBmutations had a major effect on antibiotic production by J1074, as evident from bioassays and LC-MS data. Our data support the idea thatrpoBmutations are useful tools to enhance the ability of J1074 to produce specialized metabolites.