Millet is a promising cereal with high amounts of dietary fibre and protein, and in addition, bioactive compounds with health-promoting functional properties. This study aimed to evaluate the effect of germinated and cooked whole millet flour (Pennisetum glaucum (L.) R. Br.) on protein quality, biochemical profile and intestinal health in vivo. Thirty-two male Wistar rats (21 days old) were separated into four groups, which received a casein control diet (CC; n = 8), a free protein diet (aproteic; n = 8) and two treatment diets: non-germinated millet (NM; n = 8) and germinated millet (GM; n = 8) for 29 days. The whole millet flours presented an adequate essential amino acid profile, except for lysine. The GM group presented a higher protein efficiency ratio and net protein ratio compared to the NM group. Weight gain, Lee index, and food efficiency ratio were lower in the treatment groups, compared to the control group. The GM group had lower plasma glucose, uric acid, cholesterol, and faecal pH compared to the other groups. The treatment groups presented lower triglyceride levels, higher levels of acetic and propionic acids, a larger thickness and depth of the colonic crypts, and a higher expression of PepT1 genes than the CC group. In conclusion, the millet flours demonstrated potential for controlling the lipid profile and biometric measurements. Additionally, the whole germinated millet flour provided better protein quality and improved intestinal morphology and functionality. These results indicate that the consumption of millet could be increased in human food, and considering its potential health benefits, it could be an alternative for dietary diversification, and germination is a good processing option.