ATP-binding cassette (ABC) transporters are molecular pumps ubiquitous across all kingdoms of life. While their structures have been widely reported, the kinetics governing their transport cycles remain largely unexplored. Multidrug resistance protein 1 (MRP1) is an ABC exporter that extrudes a variety of chemotherapeutic agents and native substrates. Previously, the structures of MRP1 were determined in an inward-facing (IF) or outward-facing (OF) conformation. In collaboration with the Chen Lab, we use single-molecule fluorescence spectroscopy to track the conformational changes of bovine MRP1 (bMRP1) in real time. Our results show that substrate stimulates ATP hydrolysis by accelerating the IF-to-OF transition. Importantly, the rate-limiting step of the transport cycle is the dissociation of the nucleotide-binding-domain dimer, while ATP hydrolysis per se does not reset MRP1 to the resting state. The single-molecule results guide us to determine the structure of bMRP1 under active turnover conditions, which indeed captures the transporter in its post-hydrolytic state. The combination of structural and single-molecule kinetic data illustrates how different conformations of MRP1 are temporally linked and how substrate and ATP modulate protein dynamics to achieve active transport.