Anaerobic digestion (AD) is a promising technology for stabilization of waste activated sludge (WAS) and energy recovery. Choline as a kind of alkaloid performed well in enhancing methane production from AD for WAS according to our previous research. This study furtherly investigated the influences of choline on the methanogenetic pathway and microbial community during AD process. Results showed that the biochemical methane potential was increased by 28.1% with 0.3 g/L choline applied. Significant impacts on distribution for methanogenesis pathways (acetoclastic methanogenesis (AM) and hydrogenotrophic methanogenesis (HM)) in the process were found: AM was the major pathway in the control, while HM became dominant with choline addition, accounting for 64.7% of methane production. Meanwhile, the enrichment of Methanobrevibacter (+129.2%) and Methanosarcina (+202.7%) also verified the promotion of HM pathway, indicating an increased microbial adaptability and stabilization. The analysis of choline metabolism revealed a stimulation of acidification and hydrogen production by intermediate products. © 2021 Elsevier Ltd