Secreted and integral membrane proteins compose up to one-third of the biological proteome. These proteins contain hydrophobic signals that direct their translocation across or insertion into the lipid bilayer by the Sec61 protein–conducting channel. The molecular basis of how hydrophobic signals within a nascent polypeptide trigger channel opening is not understood. Here, we used cryo–electron microscopy to determine the structure of an active Sec61 channel that has been opened by a signal sequence. The signal supplants helix 2 of Sec61α, which triggers a rotation that opens the central pore both axially across the membrane and laterally toward the lipid bilayer. Comparisons with structures of Sec61 in other states suggest a pathway for how hydrophobic signals engage the channel to gain access to the lipid bilayer.