The applicability of electric field as a pretreatment technique for inoculum preparation was confirmed in a previous study. In the present work, newly adopted electric pretreatment conditions were statistically optimized for the preparation of inoculum from anaerobic mixed cultures in dark fermentative H-2 production via response surface methodology with a Box-Behnken design. Pretreatment conditions of applied voltage, distance of electrode, and reaction time were chosen as independent variables, while H-2 yield was chosen as the response variable. Overall performance revealed that applied voltage and reaction time were slightly interdependent or significantly interactive influence on H-2 yield, and they were more influential compared with electrode distance on H-2 yield. In the confirmation test, H-2 yield of 1.42mol H-2 per mol hexose(added) was recorded, corresponding with 94.7% of the predicted response value, under applied voltage of 11V, distance of electrode of 5cm, and reaction time of 23min. Although the H-2 yield under electric pretreatment was similar with the heat pretreated yield, the energy consumption efficiency was about 40-fold lower. Therefore, it may be concluded that electric pretreatment is a potent alternate technique for inoculum preparation method in terms of energy consumption and easy applicability in the field with high effectiveness. Copyright (c) 2014 John Wiley & Sons, Ltd.