Microorganisms play a vital role in anaerobic digestion, however, bioaugmentation in a two-stage co-digestion system to directly regulate the microbial structure is limited. In this study, the different dosage of Thermoanaerobacterium thermosaccharolyticum was added in the hydrogen production stage of a two-stage anaerobic digestion system operating at a thermophilic temperature (55 °C). Results showed that Thermoanaerobacterium thermosaccharolyticum of 1.12 g had the best effect, with the cumulative hydrogen and methane yields reaching 81.54 mL/g VS and 550.98 mL/g VS respectively, which were 68.72 % and 84.45 % higher than the control group. According to the microbial analysis, the addition of Thermoanaerobacterium thermosaccharolyticum significantly changed the microbial community structure, and also its relative abundance increased in the hydrogen production stage, thereby increasing volatile fatty acids (VFAs) and hydrogen content. Although the methane production stage did not contain Thermoanaerobacterium thermosaccharolyticum, the microbial community structure changed, increasing the dominant species Anaerolineae and Limnochordia, thereby improving the efficiency of substrate decomposition and methane production. This study shows that Thermoanaerobacterium thermosaccharolyticum can promote not only hydrogen production but also the production of methane in two-stage anaerobic digestion