Improved hydrogen production from glucose was achieved by adding a specific methane inhibitor (such as chloroform) to repress the activity of methanogens in a single-chamber microbial electrolysis cells (MECs) with a double anode arrangement. A maximum hydrogen production of 8.4 +/- 0.2 mol H-2/mol-G (G represents glucose), a hydrogen production rate of 2.39 +/- 0.3 m(3) H-2/m(3)/d and a high energy efficiency (relative to the electrical input) of eta E = 165 +/- 5% had been recorded from 1 g/L glucose at a low dosage of chloroform (5 parts per thousand, v:v) and an applied voltage of 0.8 V. Almost all of the glucose was removed within 4 h, with 66% of the electrons in intermediates (mainly including acetate and ethanol), and methane gas was not detected in the MECs through 11 batch cycles. The experimental results confirmed that chloroform was an effective methane inhibitor that improved hydrogen production from glucose in the MECs. In addition, the cyclic voltammetry tests demonstrated that the electron transfer in the MECs was mainly due to the biofilm-bound redox compounds rather than soluble electron shuttles. (C) 2016, The Society for Biotechnology, Japan. All rights reserved.