In order to explore the effect of a whole-grain mixture (red rice, black rice and brown rice) on the gut microbiota and short-chain fatty acids (SCFAs) in diabetes, SCFA production was detected after in vitro fermentation and dietary intervention, and changes in the composition and abundance of the gut microbiota were analyzed by metagenomic sequencing. Compared with the blank group, the whole-grain mixture increased the abundance of the beneficial bacteria Bifidobacterium and Lactobacillus, while reducing the abundance of opportunistic pathogenic taxa. Compared with refined grains, the whole-grain mixture, rich in dietary fiber, provided more arabinose and xylose during in vitro fermentation, generating more butyric acid. Also, it increased the abundance of butyric acid-producing bacteria such as Collinsella, Parabacteroides, Anaerostipes, Oscillibacter and Faecalibacterium. Intervention with the whole-grain mixture in patients with type 2 diabetes mellitus (T2 DM) significantly increased the relative abundance of the butyric acid-producing bacteria Anaerostipes and the butyric acid level in feces, further verifying the results of in vitro fermentation. In conclusion, the whole-grain mixture positively affects the gut microbiota in T2 DM patients, promoting the production of SCFAs, especially butyric acid, which is beneficial to intestinal health.