This study was aimed to increase both the methane content and yield from rice straw by adding extra H-2 into a two-stage anaerobic digestion system. The results showed that the efficiency of H-2 utilization was 36.43%. Of the total amount of H-2 used, 47.3% was converted to CH4, thereby increasing the CH4 content and yield by 45% and 101%, respectively. This conversion was performed at a H-2/CO2 ratio of 4/1 and a recycling frequency of two times a day in an up-flow reactor (UR). Due to the different ways of CH4 production in continuously stirred tank reactor (CSTR) and UR, ethanol and acetate were produced as intermediaries, accounting for 65% and 87% of total intermediaries, respectively. Moreover, the pH values in CSTR and UR were 6.7 and 8.1, respectively. The results of the microbial community structures showed that the archaea genera of Methanosaeta and Methanobacterium were the most dominant in CSTR, whereas, Methanosaeta was the most dominant in UR. In terms of the bacterial community, Clostridia was the most abundant class in UR. Based on the results mentioned above, it is feasible to simultaneously improve both the CH4 content and yield from rice straw by adding extra H-2 into the two-stage anaerobic digestion system. (C) 2019 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.