The cometabolism as a technique for enhancing biodegradation of pentachlorophenol (PCP) has been widely used, but the effect of dosing mode of growth substrates on the cometabolic degradation of PCP in an anaerobic system with and without the addition of ZVI is still not clear. In this study, the effects of dosing modes of sodium acetate as co-substrate, i.e., one-step pulse, evenly distributed pulse, and stepwise declining pulse, and zero-valent iron (ZVI) of 1 mg l−1 on pentachlorophenol (PCP) degradation and changes in anaerobic microbial community were examined in batch tests. The results indicated that the degradation kinetic constants of PCP were related to the dosing mode of co-substrate. The complete mineralization rates of PCP in the two groups with evenly distributed pulse mode and stepwise declining pulse mode were faster than the observed rates of the group with one-step pulse mode, according to the quantitative analysis of 2,3,5,6-tetrachlorophenol, 2,3,5-tetrachlorophenol, and 2,5-chlorophenol. In addition, there was a substantial change in the content of intracellular polysaccharide in the cometabolic process of the one-step pulse and stepwise declining pulse groups. Furthermore, the electron transport system (ETS) activities reached to the maximum values of 0.841 and 0.813 ug g−1 in these two groups with the addition of 1 g l−1 ZVI, respectively. Moreover, Alcaligenes (11.02%), Comamonas (14.58%), and Ralstonia (9.16%) were the dominant genera in the groups with the three co-substrate dosing modes, respectively. This study provides a new insight into achieving anaerobic cometabolic degradation of PCP with different dosing modes of co-substrate.@en