Fermentation substrates of rice with different milling degrees (MDs) were prepared and fermented with human feces to compare their fermentation properties and effects on gut microbiota. MD 0s, MD 5s and MD 60s represented brown rice, moderately-milled rice and white rice, respectively. After in vitro fermentation, the MD 5s group showed higher starch utilization, compared with the MD 0s and 60s groups evaluated by Fourier transform infrared spectrometer, and confocal laser scanning microscope. Effects of fermentation substrates of rice with different MDs on gut microbiota were evaluated by 16S rDNA sequencing. All the sample groups reduced the pH and produced short-chain fatty acids (SCFAs) and branched-chain fatty acids. The MD 5s group exhibited higher α-diversity than the MD 0s and 60s groups. Abundances of Phascolarctobacterium, Blautia and norank_f_Ruminococcaceae were higher in the MD 0s and 5s groups, compared with the MD 60s group. These bacteria were also positively correlated with the SCFAs production via Spearman correlation analysis. In vitro culture assay revealed that fermentation substrates of MD 0s and 5s promoted the growth of two probiotics (Akkermansia muciniphila and Bifidobacterium adolescentis). Our results showed that moderate milling might be an appropriate way to produce rice products with richer nutrients and better fermentation properties.