Intrathymic T cell differentiation takes place within the thymic lobules and depends on interactions between developing thymocytes and cells of the thymic microenvironment. Along with differentiation, thymocytes migrate in an oriented progression, which is tightly regulated by a number of interactions, including one mediated by the chemokine CXCL12. It has been shown recently that SEMA-3A, a soluble member of the semaphorin family, is also involved in this human thymocyte migration and can have a chemorepulsive and de-adhesive role. Herein, we study the role of SEMA-3A on the CXCL12-driven migration of human thymocytes. We have shown that SEMA-3A is able to inhibit the chemotaxis triggered by CXCL12. Such an inhibition was seen in respect to immature and mature CD4/CD8-defined thymocyte subsets and can be reverted specifically by neutralizing anti-SEMA-3A mAb. We have also shown that SEMA-3A consistently down-regulates CXCR4 membrane expression in all CD4/CD8-defined thymocyte subsets, and this down-regulation is accompanied by a decrease in the phosphorylation of FAK and ZAP-70 protein kinases. Taken together, these results demonstrate the involvement of SEMA-3A in the regulation of CXCL12-driven human thymocyte migration, where it acts as a physiological antagonist.