In Escherichia coli and other bacteria, nickel uptake is regulated by the transcription factor NikR. Nickel binding at high-affinity sites in E coli NikR (EcNikR) facilitates EcNikR binding to the nik operon, where it then suppresses transcription of genes encoding the nickel uptake transporter, NikABCDE. A structure of the EcNikR-DNA complex suggests that a second metal-binding site is also present when NikR binds to the nik operon. Moreover, this co-crystal structure raises the question of what metal occupies the second site under physiological conditions: K+, which is present in the crystal structure, or Ni2+, which has been proposed to bind to low- as well as high-affinity sites on EcNikR. To determine which ion is preferred at the second metal-binding site and the physical basis for any preference of one ion over another in both the second metal-binding site and the high-affinity sites, we conducted a series of detailed molecular simulations on the EcNikR structure. Simulations that place Ni2+ at high-affinity sites lead to stable trajectories with realistic ion-ligand distances and geometries, while simulations that place K+ at these sites lead to conformational changes in the protein that are likely unfavorable for ion binding. By contrast, simulations on the second metal site in the EcNikR-DNA complex lead to stable trajectories with realistic geometries regardless of whether K+ or Ni2+ occupies this site. Electrostatic binding free energy calculations, however, suggest that EcNikR binding to DNA is more favorable when the second metal-binding site contains K+. An analysis of the energetic contributions to the electrostatic binding free energy suggests that, while the interaction between EcNikR and DNA is more favorable when the second site contains Ni2+, the large desolvation penalty associated with moving Ni2+ from solution to the relatively buried second site offsets this favorable interaction term. Additional free energy simulations that account for both electrostatic and non-electrostatic effects argue that EcNikR binding to DNA is most favorable when the second site contains a monovalent ion the size of K+. Taken together, these data suggest that the EcNikR structure is most stable when Ni2+ occupies high-affinity sites and that EcNikR binding to DNA is more favorable when the second site contains K+.