The role of the humic model compound, anthraquinone-2,6-disulfonate (AQDS), in the anaerobic reductive dechlorination of 2,4-dichlorophenoxyacetic acid (2,4-D) by the Fe(III)- and humic substances (HS)-reducing bacterium, Comamonas koreensis strain CY01 was investigated. The results taken as a whole indicated that (i) strain CY01 could couple glucose oxidation to 2,4-D reductive dechlorination; (ii) reductive dechlorination of 2,4-D by strain CY01 was greatly stimulated by the addition of AQDS; (iii) the transfer of electrons from biogenic AH 2QDS to 2,4-D was an abiotic process which can take place in the absence of microorganisms; and (iv) AH 2QDS was reoxidized during the chemical reaction, AQDS can serve again as electron acceptor for microorganisms, thus acting as electron shuttles. All the results suggested that 2,4-D reductive dechlorination by CY01 strain was a biochemical process that oxidizes the electron donors and transfers the electron to the acceptors through redox mediator, AQDS. We proposed the possible mechanism for the HS dependent reduction of 2,4-D. Our results suggested that microbial reduction of HS and subsequent chemical reduction of organic pollutants represent an important path of electron flow in anoxic natural environments. This work is a necessary preliminary step for better understanding the biodegradation of 2,4-D in subsurface soil.