The major efflux system in Escherichia colt is the tripartite complex AcrAB-TolC. Its homotrimeric transporter AcrB is polyspecific and extrudes antibiotics out of the bacterium. This extrusion is performed via a functional rotation, in which each monomer assumes a particular conformation. In the present study, targeted molecular dynamics simulations have been employed to obtain a molecular level understanding of the transport process. A particular focus is put on the role of water molecules in this extrusion process. It is shown that the water flows from the binding pocket toward the exit gate in the extrusion step and helps the substrate to move along this path. These results are underpinned by a detailed analysis of the electrostatic interaction energy. Furthermore, the role of water for the polyspecificity of the transporter is discussed.