The ability of a bacterial cell to monitor and adaptively respond to its environment is crucial for survival. After one- and two-component systems, extracytoplasmic function (ECF) σ factors – the largest group of alternative σ factors – represent the third fundamental mechanism of bacterial signal transduction, with about six such regulators on average per bacterial genome. Together with their cognate anti-σ factors, they represent a highly modular design that primarily facilitates transmembrane signal transduction. A comprehensive analysis of the ECF σ factor protein family identified more than 40 distinct major groups of ECF σ factors. The functional relevance of this classification is supported by the sequence similarity and domain architecture of cognate anti-σ factors, genomic context conservation, and potential target promoter motifs. Moreover, this phylogenetic analysis revealed unique features indicating novel mechanisms of ECF-mediated signal transduction. This classification, together with the web tool ECFfinder and the information stored in the Microbial Signal Transduction (MiST) database, provides a comprehensive resource for the analysis of ECF σ factor-dependent gene regulation.