BtuCD is an ABC transporter catalyzing the uptake of vitamin Bââ across the Escherichia coli inner membrane. A previously reported X-ray structure of BtuCD in complex with the periplasmic vitamin Bââ-binding protein BtuF revealed asymmetry of the transmembrane BtuC subunits. The functional relevance of this asymmetry has remained uncertain. Here we report the X-ray structure of a catalytically impaired BtuCD mutant in complex with BtuF, where the BtuC subunits adopt a distinct asymmetric conformation. The structure suggests that BtuF does not discriminate between, or impose, asymmetric conformations of BtuCD. It also explains the conformational disorder observed in BtuCDF crystals.