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.