Fibroblast growth factor 9 (FGF9) is a multifunctional polypeptide belonging to the FGF family and has functions related to bone formation, lens-fiber differentiation, nerve development, gap-junction formation and sex determination. In a previous study, we demonstrated that FGF9 stimulates the production of testosterone in mouse Leydig cells. In the present study, we used both primary mouse Leydig cells and MA-10 mouse Leydig tumor cells to further investigate the molecular mechanism of FGF9-stimulated steroidogenesis. Results showed that FGF9 significantly activated steroidogenesis in both mouse primary and tumor Leydig cells (p<0.05). Furthermore, FGF9 significantly induced the expression of phospho-Akt at 0.5 and 24 hr, phospho-JNK at 0.25, 0.5, and 24 hr, phospho-p38 at 0.5 hr, and phospho-ERK1/2 from 0.25 to 24 hr in primary Leydig cells (p<0.05). Also, FGF9 significantly up-regulated the expression of phospho-Akt at 3 hr, phospho-JNK at 0.25 hr, and phospho-ERK1/2 at 1 and 3 hr in MA-10 cells (p<0.05). Using specific inhibitors of Akt, JNK, p38, and ERK1/2, we further demonstrated that the inhibitors of Akt and ERK1/2 significantly suppressed the stimulatory effect of FGF9 on steroidogenesis in mouse Leydig cells. In conclusion, FGF9 specifically activated the Akt and ERK1/2 in normal mouse Leydig cells and the Akt, JNK and ERK1/2 in MA-10 mouse Leydig tumor cells to stimulate steroidogenesis.