Protein secretion in eukaryotes and prokaryotes involves a universally conserved protein translocation channel formed by the Sec61 complex. Unrelated small-molecule natural products and synthetic compounds inhibit Sec61 with differential effects for different substrates or for Sec61 from different organisms, making this a promising target for therapeutic intervention. To understand the mode of inhibition and provide insight into the molecular mechanism of this dynamic translocon, we determined the structure of mammalian Sec61 inhibited by the Mycobacterium ulcerans exotoxin mycolactone via electron cryo-microscopy. Unexpectedly, the conformation of inhibited Sec61 is optimal for substrate engagement, with mycolactone wedging open the cytosolic side of the lateral gate. The inability of mycolactone-inhibited Sec61 to effectively transport substrate proteins implies that signal peptides and transmembrane domains pass through the site occupied by mycolactone. This provides a foundation for understanding the molecular mechanism of Sec61 inhibitors and reveals novel features of translocon function and dynamics. Display omitted •The inhibited Sec translocon adopts a conformation optimal for substrate engagement•The inhibitor mycolactone wedges open the lateral gate of Sec61α•Mycolactone blocks the path taken by the signal peptide during engagement•Resistance mutations are likely to operate by modulating translocon dynamics Gérard et al. determine the structure of the mammalian Sec translocon in an inhibited state. Mycolactone holds the translocon in a conformation optimal for substrate engagement, wedging open the cytosolic side of the lateral gate, while also blocking the path taken by the signal peptide during engagement.