Powered by proton-motive force, the inner membrane translocase AcrB is the engine of the AcrAB-TolC efflux pump in Escherichia coli. As proton conduction in proteins occurs along hydrogen-bonded networks of polar residues and water molecules, knowledge of the protein-internal water distribution allows drawing conclusions to possible pathways of proton conduction. Here we calculated the dynamic hydration of the AcrB trans-membrane domains derived from a series of 6 x 50 ns independent molecular dynamics simulations of asymmetric AcrB embedded in a phospholipid/water environment, considering each monomer in a different protonation state. Water dynamics are different in each monomer; whereas cytoplasm and periplasm are connected by up to three different water diffusion pathways in monomer B, no such connection exists in monomer C. With zero - two connecting water ways, monomer A represents an intermediate state. We identified the residues constituting AcrB's hydrogen-bonded network and used their frequency of hydrogen bond contact to protein-internal water to determine key residues of proton conduction. We find that water flow through the trans-membrane domains is regulated by four groups of residues in a combination of side chain re-orientations and shifts of trans-membrane helices.