The metabolic pathways of protein during anaerobic digestion (AD) of waste activated sludge (WAS) were comprehensively investigated. Results showed that 100 kinds of peptidases were involved in the hydrolysis and acidogenesis processes. Serine endopeptidases (EC 3.4.21.53) and serine-type carboxypeptidases (EC 3.4.16.4) were the key enzymes of endopeptidases and exopeptidases, respectively. The pathways of ko00250 (alanine, aspartate and glutamate metabolism), ko00260 (glycine, serine and threonine metabolism), ko00270 (cysteine and methionine metabolism), ko00280 (valine, leucine and isoleucine degradation), ko00360 (phenylalanine metabolism) and ko00310 (lysine degradation) were the critical metabolic pathways of amino acids during AD of WAS, since they have complete pathways from amino acids to vital intermediates (pyruvate or acetyl-CoA). L-aspartate, L-alanine, threonine, glycine, serine, L-cysteine were the intermediate products in the conversion of protein to pyruvate, while L-leucine, L-isoleucine, phenylalanine, lysine could be directly metabolized to acetylCoA. Dechloromonas and Thauera played major roles in the crucial metabolic pathways of amino acids (ko00250, ko00260, ko00280 and ko00270). These important discoveries could provide a new biological perspective for improving AD performance.