ATP-Binding Cassette (ABC) Transporters use ATP binding and hydrolysis to power transmembrane transport of chemically diverse substrates. Current knowledge of their mechanism comes primarily from static structures of stable intermediates along the transport cycle. Recently, single-molecule fluorescence resonance energy transfer (smFRET) measurements have generated insight into the functional dynamics of transmembrane transporters, but studies to date lack direct information on the physical movement of the transport substrate. Here, we report development of an smFRET system that exploits fluorescence quenching by vitamin B12 to track its location in real time during ATP-driven transport by nanodisc-reconstituted E. coli BtuCD-F, an extensively studied type II ABC importer. Our data demonstrate that transmembrane translocation of B12 is driven by two sequential high-energy conformational changes that are inaccessible to standard structural methods because they are inherently transient. The first moves B12 from the periplasm into the transmembrane domain of the transporter; notably, this reaction is driven by hydrolysis of a single ATP molecule, in contrast to the mechanism established for several other ABC Transporter families in which ATP-binding drives the mechanochemical power-stroke prior to hydrolysis. The second mediates B12 release on the opposite side of the transporter, and it is driven by formation of a hyper-stable complex between BtuCD and BtuF. Hydrolysis of a second single ATP molecule is then required to dissociate BtuCD from the BtuF substrate-binding protein to enable it to bind B12 and initiate another round of transport. Our experiments have visualized substrate translocation in real-time at a single-molecule level and provided unprecedented information on the mechanism and dynamics of a paradigmatic transmembrane transport process.