Bacteria use diverse mechanisms to overcome antibiotics, including their destruction or chemical modification. Rifamycins, which are semi-synthetic antibiotics, adopt a characteristic basket-like structure that is essential for binding to the RNA exit tunnel of the target β-subunit of bacterial RNA polymerase to inhibit RNA synthesis. In this study, Wright and colleagues report the structure and function of a rifamycin monooxygenase (Rox) from Streptomyces venezuelae that inactivates a broad range of rifamycins. They determined the crystal structure of the Rox and identified a new molecular mechanism of resistance: monooxygenation of the C2 naphthyl ring, which results in ring opening and subsequent linearization of the antibiotic. The altered conformation prevents the antibiotic from binding to its target.