The impact of different anode acclimation methods for enhancing hydrogen production in microbial electrolysis cell (MEC) was investigated in this study. The anodes were first acclimated in microbial fuel cells using acetate, butyrate and corn stalk fermentation effluent (CSFE) as substrate before moving into MECs, respectively. Subsequently, CSFE was used as feedstock in all the three MECs. The maximum hydrogen yield with the anode pre-acclimated with butyrate (5.21 ± 0.24 L H2/L CSFE) was higher than that pre-acclimated with acetate (4.22 ± 0.19 L H2/L CSFE) and CSFE (4.55 ± 0.14 L H2/L CSFE). The current density (480 ± 11 A/m3) and hydrogen production rate (4.52 ± 0.13 m3/m3/d) with the anode pre-acclimated with butyrate were also higher that another two reactors. These results demonstrated that the anode biofilm pre-acclimated with butyrate has significant advantages in CSFE treatment and could improve the performance of hydrogen production in MEC.