The recent decline in male fertility has been linked to the increasing prevalence of endocrine disrupting chemicals that can interact with native oestrogen receptors to affect downstream signalling. There is a critical role for the cytoskeleton in facilitating spermatogenesis and Sertoli cell function, suggesting this could be a key target of oestrogenic EDCs to impact male fertility. We previously demonstrated that in human testis-derived NT2/D1 cells, exogenous oestrogen limits the bioavailability of the key testis factor SOX9 through the rapid stabilisation of microtubules, leading to a shift towards expression of ovarian developmental pathways. To further understand how oestrogen regulates the microtubule cytoskeleton, as well as actin and intermediate filaments, we performed proteomic and phosphoproteomic analyses. We show that oestrogen exposure leads to increased abundance of tubulin and hypophosphorylation of microtubule associated proteins, increased abundance of vimentin, and the rapid activation of the actin polymerising ARP2/3 complex. Together, these results demonstrate oestrogen treatment can target all three components of the cytoskeleton. Given how important the cytoskeleton is for spermatogenesis and Sertoli cell determination and function, these results provide a mechanism of how exogenous oestrogen could directly impact increasing rates of male infertility.