Although AHL-mediated quorum sensing (QS) signaling has been proved to be ecologically important in biofilm formation and aerobic granulation process, the biological role of AHL in anaerobic granule has not been experimentally investigated. In this paper, we explored the AHL level in 10 full-scale industrial anaerobic granular bioreactors and detected a total of 4 kinds of AHLs. C8-HSL and C10-HSL were indicated to be the potentially universal QS signal molecules in anaerobic granules and involved in extracellular polymeric substance (EPS) production and granulation process. The add-back experiments further supported the hypothesis that C8-HSL and C10-HSL might play an important role in facilitating anaerobic granulation through regulation of EPS synthesis. 12 Microorganisms might be regulated by AHL to play an important role in EPS production. This study provides a foundation for exploring the function of AHL in anaerobic granular systems, which may shed light on advanced AHL-based anaerobic granulation strategy.