Objective To investigate the effect and mechanism of fenvalerate (Fen) on testosterone synthesis in Leydig cells of rats testis. Methods Leydig cells of SD rat testis were isolated and purified by differential adhesion method, then treated with 0, 25, 50 and 100 μmol/L Fen for 1, 12 and 24 h, and the level of testosterone was detected by ELISA. Set blank control group(treatment with 0.1% DMSO), Fen exposure group (treatment with 100 μmol/L Fen), Fen+NAC group (treatment with 100 μmol/L Fen and 5 mmol/L NAC), Fen+CsA group (treatment with 100 μmol/L Fen and 2 mmol/L CsA), and cultured for 24 h after administration. The changes of cellular reactive oxygen species (ROS) and mitochondrial membrane potential were detected by flow cytometry, the levels of testosterone, glutathione (GSH) and cAMP were detected by ELISA, and the content of ATP was detected by chemi-luminescence, Western blotting was used to detect the expressions of superoxide dismutase (SOD), steroidogenic acute regulatory protein (StAR), 3β-hydroxysteroid dehydrogenase (3β-HSD) and cytochrome P450 cholesterol side-chain cleavage(CYP11A1). Results 100 μmol/L Fen treatment for 24 h was selected in the experiments. Compared with blank control group, the testosterone synthesis level, the contents of GSH, SOD, ATP and cAMP, mitochondrial membrane potential, and the relative expression levels of StAR, 3β-HSD and CYP11A1 decreased significantly in Leydig cells of Fen exposure group (P<0.01), the ROS content increased significantly (P<0.01). Compared with Fen exposure group, the testosterone synthesis level, the contents of GSH, SOD, ATP and cAMP, mitochondrial membrane potential, and the relative expression levels of StAR, 3β-HSD and CYP11A1 increased significantly in Leydig cells of Fen+NAC group and Fen+CsA group (P<0.05 or P<0.01), the ROS content decreased significantly (P<0.01). Conclusion Fen may cause mitochondrial damage in Leydig cells by inducing oxidative stress, resulting in the inhibition of ATP and cAMP synthesis, thereby inhibiting the expression of testosterone synthesis related proteins and enzymes dependent on cAMP/PKA signaling pathway, and ultimately leading to testosterone synthesis disorder in Leydig cells.