Abstract: Both raw and cooked millet prolamin (RFMP and CFMP) exhibited significant functions in improving glucose metabolism. Compared to RFMP, the protein quality of CFMP significantly decreased. Therefore, RFMP is more valuable as a potential food-derived hypoglycemic functional substance. In order to study the effects of RFMP on the intestinal flora and liver of type 2 diabetes (T2D) mice, C57BL/6J mice were used as the objects, and T2D models were established by high-fat diet combined with streptozotocin. The T2D mice were continuously intervened with RFMP for five weeks. Subsequently, 16S rRNA sequencing and transcriptomics technology were employed to analyze the composition of the gut microbiota and liver gene expression, respectively. The results showed that RFMP could improve glucose metabolism by alleviating the intestinal flora disorder induced by diabetes. At the genus level, RFMP intervention significantly increased the abundance of Lactobacillus, norank_f_Muribaculaceae, and Lachnospiraceae_NK4A136_group in T2D mice. Liver transcriptome results showed that the intervention of RFMP upregulated the expression levels of Fgf21, Dusp1, Rasgrp1, and HK3, while downregulated the expression level of Pck1, which were the key genes for regulating glucose metabolism. This study improved the mechanism of improving glucose metabolism with RFMP, providing a theoretical basis for the development of functional FMP product.