The X-ray crystal structure of the transporter-binding protein complex BtuCD–F, involved in the uptake of vitamin B12 across the inner membrane of Escherichia coli, is determined in an ATP analogue-bound state; the membrane-spanning BtuC subunits adopt a previously unseen conformation in which the central translocation pathway is sealed by an additional gate, and membrane transport is seen to occur through an unexpected peristaltic transport mechanism, distinct from what has been observed for other ABC transporters.