ATP-binding cassette (ABC) transporters form a large family of transmembrane importers and exporters. Using two nucleotide-binding domains (NBDs), which form a canonical ATP-sandwich dimer at some point within the transport cycle, the transporters harness the energy from ATP binding and hydrolysis to drive substrate transport. However the structural elements that enable and tune the dimerization propensity of the NBDs have not been fully elucidated. Here we compared the biochemical properties of the NBDs of human and rat TAP1, a subunit of the heterodimeric transporter associated with antigen processing (TAP). The isolated human TAP1 NBD was monomeric in solution, in contrast to the previously observed ATP-mediated homodimerization of the isolated rat TAP1 NBD. Using a series of human-rat chimeric constructs, we identified the D-helix, an α-helix N-terminal to the conserved D-loop motif, as an important determinant of NBD dimerization. The ATPase activity of our panel of TAP1 NBD constructs largely correlated with dimerization ability, indicating that the observed dimerization uses the canonical ATP-sandwich interface. The N-terminus of the D-helix from one protomer interacts with the ATP-binding Walker A motif of the second protomer at the ATP-sandwich interface. However, our mutational analysis indicated that residues farther from the interface, within the second and third turn of the D-helix, also influence dimerization. Overall, our data suggest that although the D-helix sequence is not conserved in ABC transporters, its precise positioning within the NBD structure has a critical role in NBD dimerization.