The aim of this study was to investigate the differences of pig intestinal microbes on amino acid disappearance rates of protein feed sources (soybean protein, soybean peptide and monomeric amino acid) with different molecular structure. Three about 30 kg healthy growing pigs were selected and slaughtered, 50 mL jejunum, ileum and cecal digesta were sterile collected into 500 mL anaerobic basal medium, respectively, and jejunum, ileum and cecal microbial suspensions were got by gradient centrifugation. Firstly, 50 mL anaerobic basal medium were added into 250 mL conical flask, and 5 mL jejunal microbial suspension were inoculate into 50 mL preheated anaerobic basal medium. After cultured for 4 h, 5 mL jejunal culture medium were taken for the determination of amino acids contents and enzymes activities and 5 mL ileal microbial suspension were inoculate into the above culture medium. Secondly, after cultured for another 4 h, 5 mL ileal culture medium were taken for the determination of amino acid contents and enzyme activities and 5 mL cecal microbial suspension were inoculate into the above culture medium. After cultured for another 4 h, 5 mL cecal culture medium were taken for the determination of amino acid contents and enzyme activities. The experiment consisted of 3 groups, soybean protein group (basal medium+soybean protein+microbes), soybean peptide group (basal medium+soybean peptide+microbes) and monomeric amino acid group (basal medium+monomeric amino acid+microbes), and 1 negative control group (basal medium+microbes) and 3 positive control groups (basal medium+soybean protein, basal medium+soybean peptide and basic medium+monomeric amino acids) were also consisted, and each group had 3 replicates. The results showed that the activities of lysine decarboxylase, arginine decarboxylase, histidine decarboxylase, glycine decarboxylase, tyrosine decarboxylase, glutamic-oxalacetic transaminase, glutamic-pyruvic transaminase and adenosine deaminase of protein feed sources with 3 different molecular structure were affected significantly by jejunum, ileum or cecal microbes (P<0.05 or P<0.01). The enzyme activities in monomeric amino acid group were higher overall than those in the other two groups. The enzyme activities in 3 groups were not completely consistent with the disappearance rate of amino acids. There were extremely significant differences in amino acid disappearance rates of protein feed sources with different molecular structure cultured in different intestinal microbes (P<0.01). The disappearance rate of amino acids in soybean protein group ranged from high to low:cecum, jejunum, ileum. The disappearance rate of amino acids in soybean peptide and monomeric amino acid groups with simple structure ranged from high to low:ileum, cecum, jejunum. The disappearance rate of total amino acids in soybean protein group was the highest. In conclusion, in vitro cultivation of intestinal microbes, the disappearance rate of amino acids among 3 protein feed sources with different molecular structure are different, and intestinal microbes are more active in protein decomposition of soybean.