Royal jelly (RJ) excreted by honeybees and used as a nutritional and medicinal agent has estrogen-like effects, yet the compounds mediating these effects remain unidentified. The possible effects of three RJ fatty acids (FAs) (10-hydroxy-2-decenoic-10H2DA, 3,10-dihydroxydecanoic-3,10DDA, sebacic acid-SA) on estrogen signaling was investigated in various cellular systems. In MCF-7 cells, FAs, in absence of estradiol (E(2)), modulated the estrogen receptor (ER) recruitment to the pS2 promoter and pS2 mRNA levels via only ERβ but not ERα, while in presence of E(2) FAs modulated both ERβ and ERα. Moreover, in presence of FAs, the E(2)-induced recruitment of the EAB1 co-activator peptide to ERα is masked and the E(2)-induced estrogen response element (ERE)-mediated transactivation is inhibited. In HeLa cells, in absence of E(2), FAs inhibited the ERE-mediated transactivation by ERβ but not ERα, while in presence of E(2), FAs inhibited ERE-activity by both ERβ and ERα. Molecular modeling revealed favorable binding of FAs to ERα at the co-activator-binding site, while binding assays showed that FAs did not bind to the ligand-binding pocket of ERα or ERβ. In KS483 osteoblasts, FAs, like E(2), induced mineralization via an ER-dependent way. Our data propose a possible molecular mechanism for the estrogenic activities of RJ's components which, although structurally entirely different from E(2), mediate estrogen signaling, at least in part, by modulating the recruitment of ERα, ERβ and co-activators to target genes.