Hepatocyte transplantation is a promising therapeutic approach for various liver diseases. Despite the liver's tolerogenic potential, early immune-mediated loss of transplanted cells is observed and long-term acceptance has not been achieved, yet. Patients deemed tolerant after liver transplantation presented an increased frequency of regulatory T cells (Treg ), which therefore also might enable reduction of posttransplant cell-loss and enhance long-term allograft-acceptance. We hence characterised hepatocyte-induced immune reactions and evaluated the immunomodulatory potential of Treg applying mixed lymphocyte cultures and mixed lymphocyte hepatocyte cultures. These were set up using peripheral blood mononuclear cells and primary human hepatocytes, respectively. Polyclonally expanded CD4+ CD25high CD127low Treg were added to co-cultures in single-/trans-well setups with/without supplementation of anti-interferon-gamma antibodies. Hepatocyte-induced alloresponses were then analysed by multi-colour flow-cytometry. Measurements indicated that T cell response upon stimulation was associated with interferon-gamma induced MHC class II upregulation on hepatocytes and mediated by CD4+ T cells. An indirect route of antigen presentation could be ruled out by use of fragmented hepatocytes and culture supernatants of hepatocytes. Allospecific proliferation was accompanied by inflammatory cytokine secretion. CD8+ T cells showed early up-regulation of CD69 despite lack of cell proliferation in the course of co-culture. Supplementation of Treg effectively abrogated hepatocyte-induced alloresponses and was primarily cell contact-dependent.