Heme oxygenase-1 (HO-1) is critical for the ability of immature dendritic cells (imDCs) to suppress T-cell responses. Induction of high HO-1 expression may markedly improve the tolerogenic capacity of imDCs. Here, we generated bone marrow-derived DCs (BMDCs) from BALB/c mice with low doses of GM-CSF and IL-4. The adherent BMDCs were obtained as imDCs. Upregulation of HO-1 in imDCs (HO-1(hi)-imDCs) was achieved by cobalt protoporphyrin treatment. HO-1(hi)-imDCs proved to be more maturation-resistant than conventional imDCs, with an enhanced ability to inhibit allogeneic T-cell proliferation stimulated by anti-CD3/CD28 antibodies. When donor-derived DC adoptive transfer was performed in a stringent mouse cardiac allotransplant model, the extent of graft prolongation observed with HO-1(hi) imDCs was superior to that obtained with conventional imDCs. T-cell activation and proliferation in cardiac allograft recipients was more strongly suppressed in the HO-1(hi) imDC transfusion group than that in the untreated imDC group. Furthermore, donor HO-1(hi) imDCs were able to maintain a status of high HO-1 expression and survived longer in the recipient spleens than did untreated imDCs after adoptive transfer. In vitro-generated HO-1(hi) imDCs had an enhanced tolerogenic capacity to modulate alloimmune responses both in vitro and in vivo, and thus may offer a novel antigen-specific and cost-effective strategy to induce transplant tolerance.