An electrolyte solution that includes redox couples is an essential component of a redox flow battery (RFB), and it directly affects the efficiency, power density, and cycle stability of the device. In this work, 12-phosphotungstic acid (PTA), a keggin type heteropolyacid, is evaluated as a redox active material in negative electrolyte. PTA has high redox reversibility and electrochemical activity with a kinetic constant of 0.015 cm s−1 and a low permeability of 6.17 × 10−10 cm2 min−1 through a Nafion®211 membrane. A single RFB is assembled with PTA and HI as the negative and positive redox active materials, respectively. The cell has a specific capacity of 12.8 Ah·L−1 at 100 mA cm−2; however, it exhibits poor cycle stability because of the osmotic pressure imbalance between the electrolyte solutions, the permeation of I−, and the serious hydrogen evolution side reaction. Using a mixed solution of PTA and HI as electrolytes on both sides of the cell and by adjusting the charge cutoff voltage, the cycle stability improved significantly. During 700 charge/discharge cycles at 100 mA cm−2, the battery has high coulombic efficiency of 99.6%, energy efficiency around 80.1%, and capacity retention of 99.99% per hour.