Cell therapy is a promising strategy to treat patients suffering from autoimmune or inflammatory diseases, or receiving a transplant. Our preclinical studies revealed that treatment with Autologous Tolerogenic Dendritic Cells (ATDCs) is safe in non-human primates and promotes allograft tolerance in rodents. We have now established a robust procedure for manufacturing human ATDCs, which are currently being tested in a first-in-man phase I/II clinical trial as an adjunct immunosuppressive therapy in patients undergoing kidney transplantation. In the present study, we report human ATDCs properties and their mechanisms of action on T cells.The defining characteristics of ATDCs are their suppression of T cell proliferation and their expansion of regulatory T cells through secreted factors alone. We found that ATDCs produce high levels of lactate which shape T cell responses towards tolerance. Our results showed that T cells take up ATDC-secreted lactate leading to a decreased of their glycolytic metabolism. In a humanized mouse model, ATDCs delay graft-versus-host-disease development by promoting expansion of CD4+Foxp3+ regulatory T cells, which correlates with elevated levels of lactate in the blood. The contact-independent, non-specific suppression of T cell immunity through lactate production by ATDCs is a novel mechanism of action that distinguishes ATDCs from other cell-based immunotherapies currently under clinical investigation. In light of their strong tolerogenic potential in vitro and in vivo, we believe that ATDC therapy should be extended to other clinical trials with the aim of regulating the immune response.