Gene expression in bacteria is regulated at the level of transcription initiation, a process driven by σ factors. The regulation of σ factor activity proceeds from the regulation of their cytoplasmic availability, which relies on specific inhibitory proteins called anti-σ factors. With anti-σ factors regulating their availability according to diverse cues, ExtraCytoplasmic Function σ factors (σ(ECF)) form a major signal transduction system in bacteria. Here, structure:function relationships have been characterized in an emerging class of minimal-size, transmembrane anti-σ factors, using CnrY from Cupriavidus metallidurans CH34 as a model. This study reports the 1.75 Å resolution structure of CnrY cytosolic domain in complex with CnrH, its cognate σ(ECF) and identifies a small hydrophobic knob in CnrY as the major determinant of this interaction in vivo. Unsuspected structural similarity with the molecular switch regulating the general stress response in α-proteobacteria unravels a new class of anti-σ factors targeting σ(ECF). Members of this class carry out their function via a 30-residue stretch that displays helical propensity but no canonical structure on its own.