ABSTRACT Anaerobic digestion is important for the management of livestock manure with high ammonia level. Although ammonia effects on anaerobic digestion have been comprehensively studied, the molecular mechanism underlying ammonia inhibition still remains elusive. In this study, based on metatranscriptomic analysis, the transcriptional profile of microbial community in anaerobic digestion under low (1500 mg L-1) and high NH4 + (5000 mg L-1) concentrations, respectively, were revealed. The results showed that high NH4 + concentrations significantly inhibited methane production but facilitated the accumulations of volatile fatty acids. The expression of methanogenic pathway was significantly inhibited by high NH4 + concentration but most of the other pathways were not significantly affected. Furthermore, the expressions of methanogenic genes which encode acetyl-CoA decarbonylase and methyl-coenzyme M reductase were significantly inhibited by high NH4 + concentration. The inhibition of the co-expressions of the genes which encode acetyl-CoA decarbonylase was observed. Some genes involved in the pathways of aminoacyl-tRNA biosynthesis and ribosome were highly expressed under high NH4 + concentration. Consequently, the ammonia inhibition on anaerobic digestion mainly focused on methanogenic process by suppressing the expressions of genes which encode acetyl-CoA decarbonylase and methyl-coenzyme M reductase. This study improved the accuracy and depth of understanding ammonia inhibition on anaerobic digestion.