We have previously shown that rat allogeneic DC, made immature by adenoviral gene transfer of the dominant negative form of IKK2, gave rise in-vitro to a unique population of CD4+CD25- regulatory T cells (dnIKK2-Treg). These cells inhibited Tcell response in-vitro, without needing cell-to-cell contact, and induced kidney allograft survival prolongation in-vivo. Deep insight into the mechanisms behind dnIKK2-Treg-induced suppression of Tcell proliferation remained elusive. Here we document that dnIKK2-Treg release extracellular vesicles (EV) riched in exosomes, fully accounting for the cell-contact independent immunosuppressive activity of parent cells. DnIKK2-Treg-EV contain a unique molecular cargo of specific miRNAs and iNOS, which, once delivered into target cells, blocked cell cycle progression and induced apoptosis. DnIKK2-Treg-EV-exposed T cells were in turn converted into regulatory cells. Notably, when administered in-vivo, dnIKK2-Treg-EV prolonged kidney allograft survival. DnIKK2-Treg-derived EV could be a tool for manipulating the immune system and for discovering novel potential immunosuppressive molecules in the context of allotransplantation.