Solids retention time (SRT) is a key parameter affecting the performance of anaerobic digestion. Nevertheless, the knowledge of how SRT impacts the anaerobic ecosystem is still insufficient. In this study, the shifts in system performance, microbial population, metabolic pathway, and energy metabolism induced by different SRTs (25 and 10 days) in anaerobic reactors with ethanol as the substrate were systematically investigated. Ethanol was oxidized to acetate and propionate, and methane was generated via mixotrophic pathways at SRT of 25 days due to the enrichment of ethanol oxidizers Geobacter and Pelobacter, propionate oxidizers Smithella, and methanogens Methanothrix. However, ethanol was metabolized to only acetate by Desulfovibrio which outcompeted other ethanol oxidizers and established interspecies H 2 transfer with H 2 -utilizing methanogen Methanobacterium at a SRT of 10 days. Under short SRT conditions, acetoclastic methanogens were eliminated, resulting in the accumulation of acetate and low CH 4 yield. Additionally, the major ATPase-gene owners were shifted from bacteria (F-type ATPase) to methanogens (V/A-type ATPase) with decreasing SRT and pH, suggesting the energy-metabolism turnover of anaerobic microbes when encountering different SRTs.