MsbA is an ATP-binding cassette transporter from Escherichia coli that is involved in trafficking lipid A across the inner membrane. ATP-binding cassette transporters harness the free energy of ATP binding and hydrolysis to drive the uphill translocation of substrates against their concentration gradients. A model of protein motion coupling energy input to work was inspired by crystallographic snapshots of MsbA. Central to this model is a switch in the accessibility of a transmembrane chamber, implicated in substrate binding, from an inward- to an outward-facing configuration. Here, we used spin labeling and electron paramagnetic resonance spectroscopy to systematically explore rearrangements in MsbA structure during the ATP hydrolysis cycle. Spin-label accessibility and local dynamics were determined in liposomes for the nucleotide-free intermediate and the transition state of ATP hydrolysis. The changes in the electron paramagnetic resonance parameters between these two intermediates fit a global pattern consistent with alternating access of the chamber. In the transition state of ATP hydrolysis, spin labels on the cytoplasmic side report increased dynamic restrictions and reduced water accessibility, while those on the extracellular side report increased water penetration. Furthermore, spin-label mobility and accessibility as well as their changes are consistent with those expected based on the crystal structures. The reversal in chamber hydration is likely to reduce the free energy of amphipathic substrate binding and promote translocation across the membrane.