Fibroblast growth factor 9 (FGF9) is a potent mitogen and survival factor required for morphogenesis during embryonic development and numerous biological functions at adulthood. The reproductive phenotype of mice lacking Fgf9 gene exhibits male to female sexual reversal, suggesting a crucial role of Fgf9 in male sex determination. Our previous study showed that polymorphic microsatellite of FGF9 genes is associated with 46XY female with ambiguous genitalia, implying that the aberrant expression of FGF9 might affect androgen secretion. In this study, we aimed to investigate the effect of FGF9 on testosterone production in mouse Leydig cell and to study the signalling pathways by which FGF9 modulate steroidogenesis. Our results show that mRNAs of Fgf9 and Fgfr isoforms (Fgfr2IIIc, Fgfr3 and Fgfr4) were all expressed in mouse Leydig cells. FGF9 significantly stimulates mouse Leydig cell testosterone production in a dose- and time-dependent manner. Ras-MAPK, PI3K and PKA signalling pathways are involved in the FGF9-induced steroidogenesis. These results provide supportive evidence linking the aberrant expression of FGF9 to human gonadal dysgenesis and suggest a role of FGF9 in postnatal testicular development.