Understanding antibiotic resistance mechanisms is critical for developing anti-infective therapies and genomics-based drug discovery. Yet, many knowledge gaps remain with respect to novel types of antibiotics from less explored producers such as anaerobes (clostridia). By genome editing and functional assays we found that CtaZ confers self-resistance against the copper chelator and gyrase inhibitor closthioamide (CTA) in Ruminiclostridium cellulolyticum. Biochemical analyses, bioinformatics, and X-ray crystallography revealed CtaZ as a founding member of a new subgroup of GyrI-like proteins. CtaZ is unique in binding a polythioamide scaffold in a ligand-optimized hydrophobic pocket, thus confining CTA. Genome mining using CtaZ as a handle, we discovered that previously overlooked homologs are widespread in Firmicutes, including many pathogens. In addition to characterizing a new role of a GyrI-like domain in self-resistance and unprecedented thioamide binding, this work aids uncovering related drug-resistance mechanisms.