In the present study, we aimed to evaluate the effect of UV-visible irradiation on the estrogenicity of an estrone aqueous solution by using chemical analysis associated with an in vitro bioassay and in silico analysis. An estrone aqueous solution was irradiated with an UV-visible high-pressure mercury lamp. By using the MELN in vitro cellular bioassay, based on the induction of a luciferase reporter gene upon the activation of the estrogen receptor by chemicals, we showed that the estrogenic potency of the solution increased after irradiation. High-performance liquid chromatography fractionation of the photolyzed solution followed by in vitro testing of fractions allowed the quantitation of the estrogenic potency of each fraction. Nine photoproducts were detected and characterized by liquid chromatography-mass spectrometry coupling. The observed estrogenic activity is mediated by mono- and multi-hydroxylated photoproducts; it is influenced by the position of hydroxyl groups on the steroidal skeleton. In addition, a structure-activity analysis of the hydroxylated photoproducts confirmed their ability to act as estrogen receptor ligands.