Microorganisms in anaerobic digestion (AD) are easily affected by ammonia, especially acetoclastic methanogens. Thus, in ammonia-suppressed AD systems, acetate degradation is reported to be carried out mainly by the cooperation of syntrophic acetate oxidizers and hydrogenotrophic methanogens. Previous studies have revealed ammonia inhibition on microbial flora by AD performance, but the effect mechanism of ammonia on microbial metabolism remains poorly understood. In this study, we constructed a mesophilic chemostat fed with acetate as the sole carbon source, gradually increased the total ammonia nitrogen (TAN) concentration from 1 g L to 6 g L, and employed the 16S rRNA gene, metagenomics, and metatranscriptomics analysis to characterize the microbial community structure and metabolic behavior. The results showed that even at the TAN of 6 g L (pH 7.5), the methanogenesis kept normal, the biogas production was approximately 92% of that at TAN of 1 g L and the acetate degradation ratio reached 99%, suggesting the strong TAN tolerance of the microbial community enriched. 16S rRNA gene analysis suggested that the microbial community structure changed along with the TAN concentration. predominated in methanogens all the time, in which the dominant species was gradually replaced from to with the increased TAN. Dominant bacterial species also changed and showed a significant positive correlation with increased TAN. Meta-omics analysis showed that the absolute dominant microorganisms at TAN of 6 g L were and , both of which highly expressed genes for anti-oxidative stress. and the second dominant methanogen highly expressed both acetate cleavage and CO reduction pathways, suggesting the possibility that these two pathways contributed to methanogenesis together. and some other species in Firmicutes and Synergistetes were likely acetate oxidizers in the community as they highly expressed genes for syntrophic acetate oxidization, H generation, and electron transfer. These results suggested that as well as have strong ammonia tolerance and played critical roles in acetate degradation under ammonia-suppressed conditions. The achievements of the study would contribute to the regulation and management of the AD process.