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Recent studies suggested that royal jelly and its substances may have anti-aging properties and can be utilized as nutraceuticals. However, the molecular mechanisms underlying its beneficial effects are far from clearly elucidated. Our genetic analyses suggested that the effect of royal jelly (RJ) and enzyme-treated royal jelly (eRJ) on lifespan and healthspan in Caenorhabditis elegansis modulated by the functional interplays of DAF-16, SIR-2.1, HCF-1 and 14-3-3 proteins. Previous studies have reported that DAF-16/FOXO, SIR-2.1/SIRT1, 14-3-3, and HCF-1 are important prolongevity determinants and have extensive interplays in worms and mammals. Considering the conservation of these prolongevity determinants in diverse species, this study not only provides new insights into the molecular mechanisms of royal jelly's function on healthspan promotion in C. elegans, but also has imperative implications in higher order organisms to delay aging and age-related disorders.