Biohythane is a renewable energy fuel composed of methane and hydrogen gas at a certain ratio. Microbial electrolysis cells (MECs) have been employed to produce biohythane but the composition of the produced gas is not well controlled. Herein, an innovative MEC system was developed at a large scale of 19 L to investigate biohythane production affected by operational factors. The goal was to understand the interaction between operation and performance towards the development of effective strategies for controlling biohythane composition. To achieve this goal, the performance of this MEC system was studied by varying the key operational factors including anolyte recirculation rate, external resistance, and hydraulic residence time (HRT). It was found that the optimized operational condition for this MEC system included the anolyte recirculation rate of 800 mL min(-1), external resistance of 1 Omega, and HRT of 24 h. This condition led to the biohythane production of 0.64 +/- 0.06 L day(-1) with 16.5% H-2 proportion and positive net energy recovery of 1.52 +/- 0.19 kWh day(-1). The ANOVA test indicated that the anolyte recirculation rate significantly impacted the methane production rate while the external resistance strongly affected the proportion of hydrogen gas in biohythane. HRT had a minor effect on the biohythane composition but could significantly influence organic removal rate. This is the first study that attempted to use operational factors to control biohythane composition, and its results will provide important implications to formulate control strategies for biohythane production and to scale up MEC systems towards practical applications.