Recent loss-of-function and gain-of-function studies have revealed that transcription factor Sox9 is required for testis formation by governing Sertoli cell differentiation, and thereafter regulating transcription of Sertoli marker genes. In the present study, we identified a novel isoform of Vinexin, which is expressed in somatic cells but not germ cells of sexually indifferent stages of fetal gonads. After the sex is determined, the expression continues in testicular Sertoli cells. Immunohistochemical analyses with a specific antibody to Vinexin indicated that Vinexin gamma is localized in the cytoplasm. Functional studies with C3H10T1/2 cells showed that Vinexin gamma acted as a scaffold protein to activate MEK and ERK through interaction with c-Raf and ERK. Ultimately, Sox9 transcription was induced by Vinexin gamma. This up-regulation of Sox9 expression disappeared when the cells were treated with a specific MEK inhibitor, U0126. To determine the role of Vinexin gamma during gonad formation, the gene was disrupted by targeted mutagenesis. The phenotype displayed by the mice indicated that ERK activation was decreased in the Vinexin gamma(-/-) XY gonads, and Sox9 expression was down-regulated. Thus, Vinexin gamma seems to be implicated in regulation of Sox9 gene expression by modulating MAPK cascade in mouse fetal gonads.