Timely negative regulation of the immune system is critical to allow it to perform its duty while maintaining it under tight control to avoid overactivation. We previously reported that the neuronal receptor neuropilin-1 (NP-1) is expressed in human lymph nodes. However, the role of NP-1 interaction with its physiological ligand semaphorin-3A (Sema-3A) on immune cells remains elusive. Here we show that Sema-3A is expressed by activated DC and T cells, and that its secretion in DC/T cell cocultures is delayed. Sema-3A/NP-1 interaction down-modulated T cell activation since addition of Sema-3A in DC/T cell cocultures dramatically inhibited allogeneic T cell proliferation. More importantly, neutralization by blocking antibodies or by antagonist peptide of endogenous Sema-3A produced by DC/T cell cocultures resulted in a 130% increase in T cell proliferation. Sema-3A acted directly on T cells, since it could block anti-CD3/CD28-stimulated proliferation of T cells. Finally, immunomodulatory functions of Sema-3A relied on the blockage of actin cytoskeleton reorganization, affecting TCR polarization and interfering with early TCR signal transduction events such as ZAP-70 or focal adhesion kinase phosphorylation. Therefore, we propose that Sema-3A secretion and the resulting NP-1/Sema-3A interaction are involved in a late negative feedback loop controlling DC-induced T cell proliferation.