Nuclear steroid hormone receptors are ubiquitously expressed transcription factors whose activity can be altered by post-translational modifications, such as phosphorylation. The consequences of post-translational modifications have been described for several members of the nuclear steroid hormone receptor superfamily; however, little is known about the effects of oestrogen receptor (ER)β phosphorylation in the brain. Moreover, to our knowledge, the presence of phosphorylated ERβ has not been detected in the brain of any species to date. Oestrogen receptor β is highly expressed in several regions of the brain and in vitro studies have demonstrated that it can be phosphorylated at two serine residues (S87 and S105) in the N-terminal AF-1 region. The present study aimed to determine whether phosphorylated ERβ is detectable in the hippocampus of aged female rats, as well as the functional consequences of ERβ S87 and S105 phosphorylation on transcriptional activity in neuronal cells. First, we used a novel PhosTag™ approach to detect phosphorylated forms of ERβ in the dorsal hippocampus of aged female rats. The data obtained demonstrated abundant forms of phosphorylated ERβ in the dorsal hippocampus, suggesting that this post-translational modification might be an important regulator of ERβ function. To assess the functional consequences of ERβ phosphorylation in neuronal cells, we created phospho-mimetic (S87E, S105E) and phospho-null (S87A, S105A) ERβ receptors that were transiently transfected in a hippocampal-derived cell line. Collectively, our results showed that phosphorylation of S87 and S105 altered both ligand-independent and ligand-dependent ERβ transcriptional regulation. Overall, these data demonstrate that phosphorylated forms of ERβ are present in the brain of aged female rats and that phosphorylation of ERβ could differentially alter ERβ-mediated gene expression.