Sertoli cells (SCs) are highly differentiated epithelial cells which play an essential role in the functional development of the testis and hence in the expression of the male phenotype. Sertoli cells form the blood-testis barrier making an extraordinary microenvironment where male germ cells develop and are under strict hormonal control. Sertoli cells express the cytokine glial cell line-derived neurotrophic factor (GDNF) under control of the follicle-stimulating hormone. GDNF is an essential regulator of SCs self-renewal and survival in vitro and is required for maintenance of the undifferentiated spermatogonial stem cells population in vivo. Various drugs, particularly alkylating agents, have been shown to be gonadotoxic. Methotrexate (MTX) is an anti-metabolite widely used in the treatment of neoplastic disorders, rheumatoid arthritis and psoriasis. The present study explored the mechanism of cytotoxic and genotoxic effects of MTX on a primary culture of Sertoli cells in vitro. DNA damage was evaluated using the Comet assay and the cell death was identified as apoptosis using terminal deoxynucleotidyl transferase- (TdT-) mediated dUTP nick end labeling (TUNEL) assay. mRNA expression and proteins levels of GDNF, P53 and ataxia telangiectasia mutated (ATM) were also investigated using quantitative polymerase chain reaction (qPCR) and Western blot methods. Results of the present study clearly showed that MTX -induced DNA damage as evident from the different Comet assay parameters. The GDNF disruption seen in our in vivo experiments correlates with the sudden increase of activation of p53 and ATM in Sertoli cells. These results imply that MTX affects the Sertoli cells, inducing GDNF proteins. This disruption signifies the loss of some support mechanism for spermatogenic cells and could be the cause of the increased apoptotic cells.