The NarK nitrate/nitrite antiporter imports nitrate (a mineral form of the essential element nitrogen) into the cell and exports nitrite (a metabolite that can be toxic in high concentrations) out of the cell. However, many details about its operation mechanism remain poorly understood. In this work, we performed steered molecular dynamics simulations of the anion translocations and quantum-chemistry model calculations of the binding sites to study the wild-type NarK protein and its R89K mutant. Our results confirm the importance of the two strictly conserved binding-site arginine residues (R89 and R305) and two glycine-rich signature motifs (G164–M176 and G408–F419) in anion’s movement through the pore. However, we have not observed large-amplitude global conformational changes of the protein during the anion migration, deviating from traditional models that require such. For the R89K mutant, we find that the anion, especially nitrite, can be slowed down or trapped in the pore due to its competition for a proton with the lysine. Our findings provide a possible explanation on why the arginine-to-lysine mutation, despite preserving the charge, impedes or abolishes the anion transport in such mutants of NarK and other similar nitrate/nitrite exchangers.