This study examined the bioavailability and bioactivity of selenium (Se) from staple cereals, wheat, pearl millet, and maize, in Se-deficient rats (Wistar strain (OUT-Wister, IND-cft (2c)). The bioavailability and bioactivity of Se were determined by measuring the Se contents of the tissue and organs and activities of Se-dependent enzymes. Se-deficient rats were repleted with Se through wheat, pearl millet, and maize. The wheat diet exhibited the highest bioavailability of Se, followed by pearl millet and maize. The bioactivity of Se, as indicated by the activity of the Se-dependent enzymes, was found to be significantly ( p < 0.001) higher in the organs of rats fed the wheat diet, followed by pearl millet and maize diets. The deficiency of Se resulted in a significant decrease ( p < 0.001) in the activity of antioxidant enzymes in circulation and organs. The staples wheat, pearl millet, and maize have a high bioavailability of Se.