Extracorporeal photopheresis (ECP) may represent an alternative to immunosuppression, as a means of reducing rejection after thoracic organ transplantation. The mechanism by which ECP exerts its protective effects has, until now, remained elusive. We analyzed peripheral blood mononuclear cells of four children with chronic heart and lung transplant rejection, who received ECP in addition to conventional immunosuppressive treatment. The effects of ECP were evaluated at each cycle, comparing blood samples from the same patient collected before and after treatment. In vitro, peripheral blood mononuclear cells treated with ECP undergo apoptosis and are phagocytosed by immature dendritic cells, which, in turn, acquire a tolerogenic phenotype. The frequency of T cells, with a regulatory phenotype and strong suppressive activity, was significantly increased in the blood of ECP-treated patients. The immunomodulatory effects of ECP may be explained by its ability to increase the frequency of regulatory T cells with inhibitory action on transplant immune rejection.