D-amino acids (D-AAs) have various biological activities, such as activation of N-methyl-D-aspartic acid (NMDA) receptor as a co-agonist by D-Ser. Since several free D-AAs are released in the broth monocultured with bacterium and D-AAs are probably utilized for bacterial communication, we presume that intestinal microbiota releases several kinds of free D-AAs, which may be involved in the hosts' health. However, presently, only four free D-AAs have been found in the ceacal lumen, but not in the colonic lumen. Here, we showed, by simultaneous analysis of chiral AAs using high-sensitivity liquid chromatography-tandem mass spectrometry (LC-MS/MS), that 12 free D-AAs (D-Ala, D-Arg, D-Asp, D-Gln, D-Glu, D-allo-Ile, D-Leu, D-Lys, D-Met, D-Phe, D-Ser, and D-Trp) are produced by intestinal microbiota and identified bacterial groups belonging to Firmicutes as the relevant bacterial candidates.