The nuclear localisation of SOX9 is essential for Sertoli cell differentiation and subsequent development of the testis. Exogenous oestrogen exposure of Sertoli cells can cause cytoplasmic retention of SOX9, leading to upregulation of key ovarian markers. The MAPK pathway can promote or inhibit SOX9 or b-catenin to tilt the balance between testis and ovarian genes in somatic cells. Thus, the MAPK pathway presents as a potential target of oestrogen to impact SOX9. Furthermore, the MAPK pathway and a key MAPK, ERK1/2, is known to affect the microtubule network. Cytoplasmic SOX9 requires a stabilised microtubule network, therefore we hypothesised that oestrogen could stabilise microtubules via ERK1/2 to promote cytoplasmic retention of SOX9. We treated the human testis cell line NT2/D1 with oestrogen for 30 minutes in the presence or absence of the ERK1/2 inhibitor U0126 and examined SOX9, tubulin and phosphorylated ERK1/2 by immunofluorescence. Oestrogen rapidly blocked the nuclear translocation of SOX9 in NT2/D1 cells and the microtubule network appeared stabilised. Phosphorylated ERK1/2 was more abundant and localised in the nucleus following oestrogen treatment. This effect was reduced upon pre-treatment with the ERK1/2 inhibitor U0126. Overall, these data suggest that oestrogen can rapidly activate ERK1/2 to stabilise microtubules and cause cytoplasmic retention of SOX9. We have revealed a previously unknown mechanism for oestrogen in impacting the function and differentiation of Sertoli cells.