Microbial desalination cell (MDC) is a promising technology to desalinate water and generate electrical power simultaneously. The objectives of this study were to investigate the desalination performance of monovalent and divalent cations in the MDC, and discuss the effect of ion characteristics, ion concentrations, and electrical characteristics. Mixed salt solutions of NaCl, MgCl₂, KCl, and CaCl₂ with the same concentration were used in the desalination chamber to study removal of cations. Results showed that in the mixed salt solutions, the electrodialysis desalination rates of cations were: Ca²⁺ >Mg²⁺>Na⁺>K⁺. Higher ionic charges and smaller hydrated ionic radii resulted in higher desalination rates of the cations, in which the ionic charge was more important than the hydrated ionic radius. Mixed solutions of NaCl and MgCl₂ with different concentrations were used in the desalination chamber to study the effect of ion concentrations. Results showed that when ion concentrations of Na⁺ were one-fifth to five times of Mg²⁺, ion concentration influenced the dialysis more profoundly than electrodialysis. With the current densities below a certain value, charge transfer efficiencies became very low and the dialysis was the main process responsible for the desalination. And the phosphate transfer from the anode chamber and potassium transfer from the cathode chamber could balance 1%–3% of the charge transfer in the MDC.