RegIIIα is an antibacterial protein that forms hexametric pores on Gram-positive bacterial membranes leading to cell lysis, however, hexamers can further assemble into filaments diminishing RegIIIα activity. Here, the authors use cryo-electron microscopy to reveal the 2.2 Å structure of RegIIIα filaments, uncovering a distinct subunit orientation and key interfaces for RegIIIα assembly, proposing the inhibitory mechanism of the pro-segment of RegIIIα.