Abstract: The gut microbiota modulation abilities of whole wheat grain (WW) and refined wheat grain (RW) were studied in vitro. In the experiment, the WW and RW were firstly digested in vitro, and then the digesta was fermented by human fecal flora, and the new generation of 16S rRNA sequencing technology was used to analyzed the microbial structure. Results:Compared with RW, the contents of dietary fiber and phenolic compounds in WW was significantly increased (p<0.05), and was 1.49 times and 1.45 times of RW, respectively. On the composition of intestinal microbes, fermentation of WW and RW all reduced the diversity of intestinal microorganisms, but significantly increased the contents of short-chain fatty acids in the fermentation broth (p<0.05), and at the same time regulated the species composition of the intestinal flora, the relative abundance of Bacteroidetes decreased, thick-walled bacteria relative abundance increased. Compared with RW, the WW increased the relative abundance of Aminoococcus, Bifidobacterium, Marchantia, Megaomonas and Megasococcus, and the relative abundance of probiotic-Bifidobacterium was 1.19 times of fermented RW, but decreased the relative abundance of Desulfovibrio, Bacteroides, Echinococcus, Faecalibacterium, Pasteurella and Sutterella, especially pathogenic-Faecalibacterium, and its relative abundance was reduced by 52%.