ATP-binding cassette (ABC) transporters constitute one of the largest superfamilies of membrane proteins that translocate variable substrates through the transmembrane domains (TMDs) powered by the ATP-driven nucleotide-binding domain (NBDs) engines. The coupling helices (CHs) located at the NBD-TMD interfaces play important roles in the structural transition between the inward- and outward-facing conformations. Here, we review our recent studies on the roles of CHs and the effects of ATP and substrates in the functional dynamics of ABC transporters.