Influence of different pretreatment methods applied on anaerobic mixed inoculum was evaluated for selectively enriching the hydrogen (H2) producing mixed culture using glucose as substrate. The cumulative H2 yield and H2 production rate were found to be dependent on the type of pretreatment procedure adopted on the parent inoculum. They could be increased by appropriate pretreatment methods, including use of heat, alkaline or acidic conditions. Along with the processing temperature and time of heat pretreatment and alkaline of alkali pretreatment increasing, the H2 yield increased and then declined, but it declined and then increased as the acidity of acid pretreatment increasing. Among the studied pretreatment methods, the heat pretreatment methods procedure enabled higher H2 yield and the maximum H2 production rate, then were alkali and acid pretreatment methods. When the inoculum was heat-treated at 80°C for 30 min, the highest cumulative H2 yield was obtained at 2152.0 mL, which was 53.20% higher than the control, and the maximum H2 production rate was 178.0 mL h-1, which was 122.0% higher than that of the Ctrl (138.0mL h-1).