Abstract BtuF is the periplasmic binding protein (PBP) that binds vitamin B 12 and delivers it to the periplasmic surface of the ABC transporter BtuCD. PBPs generally exhibit considerable conformational changes during ligand binding process, however, BtuF belongs to a subclass of PBPs that, doesn't show such behavior on the basis of the crystal structures. Employing steered molecular dynamics on the B 12-bound BtuF, we investigated the energetics and mechanism of BtuF. A potential of mean force along the postulated vitamin B 12 unbinding pathway was constructed through Jarzynski's equality. The large free energy differences of the postulated B 12 unbinding process suggests the B 12-bound structure is in a stable closed state and some conformation changes may be necessary to the B 12 unbinding. From the result of the principal component analysis, we found the BtuF-B 12 complex shows clear opening-closing and twisting motion tendencies which may facilitate the unbinding of B 12 from the binding pocket. The intrinsic flexibility of BtuF was also explored, and it's suggested the Trp44-Gln45 pair, which is situated at the mouth of the B 12 binding pocket, may act as a gate in the B 12 binding and unbinding process.