Bacterial-derived polyketide and non-ribosomal peptide natural products are crucial sources of therapeutic agents and yet little is known about the conditions that favor activation of natural product genes or the regulatory machinery that controls their transcription. Recent findings suggest that the σ54 system, which includes σ54-loaded RNA polymerase and transcriptional activators called enhancer binding proteins (EBPs), might be a common regulator of natural product genes. Here, we explore this idea by analyzing four putative σ54 promoters identified in the sequences of Myxococcus xanthus natural product gene clusters. We show that mutations in the putative σ54-RNA polymerase binding regions reduce in vivo promoter activities during growth and development. We also show that the EBP Nla28 is important for the in vivo activities of three natural product promoters, that Nla28 binds to wild-type fragments of these promoters in vitro, and that in vitro binding is lost when the putative Nla28 binding sites are mutated. These results indicate that the natural product promoters are bona fide σ54 promoter elements and three are direct targets of Nla28. Interestingly, the vast majority of experimentally confirmed and putative σ54 promoters in M. xanthus natural product clusters are located within genes and not in intergenic sequences.