To investigate substrate recruitment and transport across the Escherichia coli Ammonia transporter B (AmtB) protein, we performed molecular dynamics simulations of the AmtB trimer. We have identified residues important in recruitment of ammonium and intraluminal binding sites selective of ammonium, which provide a means of cation selectivity. Our results indicate that A162 guides translocation of an extraluminal ammonium into the pore lumen. We propose a mechanism for transporting the intraluminally recruited proton back to periplasm. Our mechanism conforms to net transport of ammonia and can explain why ammonia conduction is lost upon mutation of the conserved residue D160. We unify previous suggestions of D160 having either a structural or an ammonium binding function. Finally, our simulations show that the channel lumen is hydrated from the cytoplasmic side via the formation of single file water, while the F107/F215 stack at the inner-most part of the periplasmic vestibule constitutes a hydrophobic filter preventing AmtB from conducting water.