Among its most crucial functions as a key regulator of testis development, SOX9 drives the differentiation of Sertoli cells from the somatic cell population of the primitive gonad. In turn, SOX9 activity within Sertoli cells plays a vital role in the differentiation of several other testicular cell lineages and morphogenesis of the testis. In this study, we aimed to design an in vitro assay to model molecular mechanisms regulated by SOX9 in developing human Sertoli cells. Firstly we aimed to improve transfection efficiency to ensure sensitivity of the assays. We found that NT2/D1 transfection efficiency can be improved from below 20% to over 40% by increasing cell density and including a starvation step. qRT-PCR analysis of NT2/D1 cells transfected with SOX9-overexpression plasmid showed that when compared to empty vector, gene expression of SOX9 itself was significantly increased. In contrast, SOX9 transcript levels were downregulated by 46% in SOX9 knockdown cells compared to the control transfected cells. Cells with reduced SOX9 expression showed a significantly reduced rate of adhesion (P<0.05) and proliferation (P<0.05) compared to control. When SOX9 expression was reduced, there was no change in rate of migration of NT2/D1 cells compared to control. To test if SOX9 controls the establishment of polarity in NT2/D1 cells, transepithelial electrical resistance assays were used. SOX9 knockdown resulted in significantly reduced transepithelial resistance compared to control (P<0.01). Immediately after resistance reading were taken, the inserts were fixed and immunofluorescence was carried out to investigate the possible contribution of two markers of Sertoli cell polarity in the developing testis, Laminin and N-Cadherin. Laminin showed weaker expression, while N-Cadherin expression was lost. In conclusion, we have developed a human cell model where SOX9’s role in critical Sertoli cell functions (proliferation, migration, adhesion and polarity) can be investigated.