In mammals, male sex determination starts when the Y chromosome Sry gene is expressed within the undetermined male gonad. One of the earliest effect of Sry expression is to induce up-regulation of Sox9 gene expression in the developing gonad. SOX9, like SRY, contains a high mobility group domain and is sufficient to induce testis differentiation in transgenic XX mice. Before sexual differentiation, SOX9 protein is initially found in the cytoplasm of undifferentiated gonads from both sexes. At the time of testis differentiation and anti-Müllerian hormone expression, it becomes localized to the nuclear compartment in males whereas it is down-regulated in females. In this report, we used NIH 3T3 cells as a model to examine the regulation of SOX9 nucleo-cytoplasmic shuttling. SOX9-transfected cells expressed nuclear and cytoplasmic SOX9 whereas transfected cells treated with the nuclear export inhibitor leptomycin B, displayed an exclusive nuclear localization of SOX9. By using SOX9 deletion constructs in green fluorescent protein fusion proteins, we identified a functional nuclear export signal sequence between amino acids 134 and 147 of SOX9 high mobility group box. More strikingly, we show that inhibiting nuclear export with leptomycin B in mouse XX gonads cultured in vitro induced a sex reversal phenotype characterized by nuclear SOX9 and anti-Müllerian hormone expression. These results indicate that SOX9 nuclear export signal is essential for SOX9 sex-specific subcellular localization and could be part of a regulatory switch repressing (in females) or triggering (in males) male-specific sexual differentiation.