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Objective: This study was to investigate the effect of annexin A5 on testosterone secretion from primary rat Leydig cells and the underlying mechanisms.Methods: Isolated rat Leydig cells were treated with annexin A5.Testosterone production was detected by chemiluminescence assay.The protein and mRNA of steroidogenic acute regulatory(StAR),P450scc,3β-hydroxysteroid dehydrogenase(3β-HSD),17β-hydroxysteroid dehydrogenase(17β-HSD)and 17α-hydroxylase were examined by western blotting and RT-PCR,respectively.Results: Annexin A5 significantly stimulated testosterone secretion from rat Leydig cells in dose-and timedependent manners and increased mRNA and protein expression of StAR,P450scc,3β-HSD and 17β-HSD but not 17α-hydroxylase.Annexin A5 knockdown by siRNA significandy decreased the level of testosterone and protein expression of P450scc,3β-HSD and 17β-HSD.The significant activation of ERK1/2 signaling was observed at 5,10,and 30 min after annexin A5 treatment.After the pretreatment of Leydig cells with ERK inhibitor PD98059(50 μmol/L)for 20 min,the effects of annexin A5 on promoting testosterone secretion and increasing the expression of P450scc,3β-HSD and 17β-HSD were completely abrogated(P<0.05).Conclusions: Thus,ERK1/2 signaling is involved in the roles of annexin A5 in mediating testosterone production and the expression of P450scc,3β-HSD and 17β-HSD in Leydig cells.