Abstract: Objective To investigate the effects of Equol on genes and protein expression of testosterone synthesis related enzymes and Vimentin in testis of perinatal mice in vitro. Methods Testes were isolated and cultured in infiltrating type rotating device for 72 h. The testes were randomly divided into five groups and treated with Equol (DMSO control, 0.01, 0.10, 1.00, 10.00 μmol/L Equol). Morphological changes were observed by HE staining under optical microscope. Expressions of 3β-hydroxysteroid dehydrogenase (3β-HSD),P450 side-chain cleavage enzyme (P450scc), Vimentin were detected by real-time PCR and immunohistochemistry. Results No apparent morphological change in testes was observed compared to control group. The mRNA expression of 3β-HSD, P450Scc, Vimentin show no statistical significance(P>0.05) in all Equol group, while the protein expressions of 3β-HSD, Vimentin, P450scc increased in 0.10 μmol/L and decreased in 10.00 μmol/L Equol group. Conclusion Equol exposure can affect 3β-HSD, P450Scc, Vimentin expression in testes in vitro, means Equol may have potential adverse effects on testosterone production and spermatogenesis.