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Microbial desalination cells are considered a low-energy-consumption, clean technology to simultaneously purify wastewater and desalinate saline water by utilizing the in situ energy source contained in wastewater. To enhance desalination performance and achieve an optimal membrane stack configuration, an enlarged stacked microbial desalination cell (SMDC) has been developed and tested with 6–14 desalination cells. The cross-membrane area of the enlarged SMDC is 100 cm 2 . The anode and cathode volumes are both 200 mL. To reduce internal resistance, the width of desalination cells is kept as <0.5 mm. The optimal configuration with 10 desalination cells achieves the highest total desalination rate (TDR) of 423 mg/h and the highest charge transfer efficiency (CTE) of 836% when treating the 20 g/L NaCl solution. During this process, the junction potential across membranes increases from 0 to 374 mV, and occupies up to 74% of the total potential loss inside the SMDC. This shows that the SMDC used in this work achieves the highest TDR and CTE among the reported studies, and the junction potential should be effectively controlled to achieve the desired desalination performance in future practical applications. [ABSTRACT FROM AUTHOR]