Estradiol (E2) is a well-known hormone in vertebrates whereas in invertebrates its unambiguous presence was verified only in some species. Weather this presence is also associated to similarly conserved roles in animal phylogeny is similarly uncertain. Due to their phylogenetic position, echinoderms represent ideal experimental models to provide evolutionary insights into estrogen appearance and function. Therefore, in this research, we investigated if E2 is truly present and has a role in the reproductive biology of the sea urchin Paracentrotus lividus. Presence of 17β estradiol in body fluids was confirmed by liquid chromatography–mass spectrometry. By immunological methods (RIA) we evaluated the physiological circulating E2 levels of adult specimens and, on the basis of these, we directly administered E2 to study its metabolism and its putative effects on gonad development at physiological doses. Although different E2 tested concentrations, a correspondent dose-dependent increase of hormone levels was not found in both body fluids and gonads, suggesting the presence of potent homeostatic/detoxification mechanisms. These latter do not involve enzymes such as aromatase-like, sulfotransferase-like and acyltransferase-like, whose activities were not affected by E2 administration. Despite the increase of endogenous E2, the treatment did not induce significant variations in none of the considered reproductive parameters. Overall, this research (1) provides definitive evidence of E2 presence in sea urchin tissues and (2) demonstrate that, differently from vertebrates and starfish, E2 does not play a key role in sea urchins reproductive processes. Intra-phylum differences suggest the existence of class-specific hormonal mechanisms and highlight the risk of Phylum generalization.