Royal jelly (RJ) and selenium (Se)-rich foods have well-known health benefits which are attributable to a broad range of pharmacologic effects including antioxidant, bacteriostatic, anticancer, and immunoregulatory activities. However, there was no study to combine Se with RJ. Here, Se-rich RJ (SRJ) was produced by feeding sodium selenite to honeybees (Apis mellifera). To further clarify the function of SRJ, mice were then fed RJ or SRJ for 30 days, and their antioxidant capacity and gut microbiota profile were analyzed. The results showed that SRJ treatment could more effectively increase glutathione peroxidase levels in the liver and kidney, as well as total antioxidant activity in the liver and superoxide dismutase level in the kidney. Additionally, the ratio of Firmicutes/Bacteroidetes and relative abundance of the Lachnospiraceae and Prevotellaceae families were increased, whereas the abundance of Helicobacterceae was decreased in mice treated with SRJ. At the genus level, SRJ increased the relative abundance of Lachnospiraceae NK4A136 group, Prevotellaceae UCG 001, Rikenellaceae RC9 gut group, and Oscillibacter and decreased that of Alistipes. And the functional prediction of gut microbiota indicated SRJ treatment could enhance the amino acid metabolism. Correlation analysis indicated that SRJ could optimize the functional network of gut microbiota and the interactions between the gut microbiota and the host. These results suggested the SRJ had potential therapeutic applications in the improvement of overall health or treatment of diseases related to oxidative stress or dysbiosis.