Microbial electrolysis cells (MECs) for hydrogen production exhibit great advantages over many other biohydrogen production techniques in terms of versatility of substrate and hydrogen yield. However, hydrogen and acetate scavenging by methanogens puts forward a great challenge to the application of single chamber MECs when using mixed culture. In this study, we investigated the feasibility of using acetylene, a low-cost fuel and chemical building block, to selectively inhibit methanogenesis in single chamber MECs. Results demonstrate that the periodical injection of low concentration acetylene (1% and 5%) can successfully inhibit methanogenesis in MECs using both acetate and glucose as substrates. Current generation by exoelectrogens and the syntrophy between fermentative bacteria and exoelectrogens, however, were not negatively affected. Compared with the classic methanogen inhibitor, 2-Bromoethanesulfonate (BES), the low concentration acetylene demonstrates superior effectiveness in MECs. These results demonstrate the great potential of using acetylene as a cost-effective inhibitor against methanogenesis in MECs.