Microbial extracellular electron transfer (EET) drives various globally important environmental phenomena and has biotechnology applications. Diverse prokaryotes have been proposed to perform EET via surface-displayed "nanowires" composed of multi-heme cytochromes. However, the mechanism that enables only a few cytochromes to polymerize into nanowires is unclear. Here, we identify a highly conserved omcS-companion (osc) cluster that drives the formation of cytochrome OmcS nanowires in Geobacter sulfurreducens. Through a combination of genetic, biochemical, and biophysical methods, we establish that prolyl isomerase-containing chaperon OscH, channel-like OscEFG, and β-propeller-like OscD are involved in the folding, secretion, and morphology maintenance of OmcS nanowires, respectively. OscH and OscG can interact with OmcS. Furthermore, overexpression of oscG accelerates EET by overproducing nanowires in an ATP-dependent manner. Heme loading splits OscD
ΔoscD accelerates cell growth, bundles nanowires into cables. Our findings establish the mechanism and prevalence of a specialized and modular assembly system for nanowires across phylogenetically diverse species and environments.