Abstract: Objective To investigate the effects of DEHP in testosterone synthesis and the related genes expression in the fetal testis of male mouse by organ culture in vitro. Methods The testis tissues were cultured in infiltrating type rotating device for 72 hours. The culture and gas were changed every 24 hours. The testis tissues were divided into the DMSO control group and four DEHP groups (the terminal concentration were 0.1, 1.0, 10.0, 100.0 μmol/L). The testosterone levels in the cultured medium were measured by radioimmunoassay, the INHBβ levels were measured by Elisa, the gene expressions related with testosterone synthesis were detected by real-time PCR, the morphological changes of cultured testis were observed by HE staining under optical microscope, the expressions of related proteins were measured by immunohistochemistry. Results Compared to the control group, the levels of testosterone synthesis in 0.1, 1.0, 10.0 μmol/L groups were increased, but decreased in 100.0 μmol/L group which were exposed in DEHP for 48 hours and 72 hours. There was an increase of INHBβ synthesis in 0.1 and 1.0 μmol/L groups, but a decrease in 10.0 and 100.0 μmol/L groups. The gene expressions of 3β-HSD, P450c17, P450Scc, vimentin were significantly decreased compared to that of control group especially in 1.0 and 10.0 μmol/L groups (P<0.05), but the expressions of INHBβ had no significant changes. There were no apparent morphological changes in testis tissue by HE staining. A significant increase of the three proteins (3β-HSD, P450c17, P450Scc) and a significant decrease of vimentin were observed in 10.0 and 100.0 μmol/L groups (P<0.05). Conclusion DEHP exposure can affect the testosterone synthesis in the fetal testis of male mouse. The regulation of gene expression involving in testosterone synthesis might be the mechanism.