H2 can be obtained from glucose by fermentation at mesophilic temperatures, but here we demonstrate that hydrogen can also be obtained from glucose at low temperatures using microbial electrolysis cells (MECs). H2 was produced from glucose at 4°C in single-chamber MECs at a yield of about 6mol H2mol-1 glucose, and at rates of 0.25±0.03-0.37±0.04m3 H2m-3d-1. Pyrosequencing of 16S rRNA gene and electrochemical analyses showed that syntrophic interactions combining glucose fermentation with the oxidization of fermentation products by exoelectrogens was the predominant pathway for current production at a low temperature other than direct glucose oxidization by exoelectrogens. Another syntrophic interaction, methanogenesis and homoacetogenesis, which have been found in 25°C reactors, were not detected in MECs at 4°C. These results demonstrate the feasibility of H2 production from abundant biomass of carbohydrates at low temperature in MECs. © 2012 Elsevier Ltd.