Background. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature cells that suppress immune responses during organ transplantation and participate in mediating long-term graft survival and immune tolerance in animal transplant models. However, their role in regulating transplant tolerance in human subjects is not well understood. In the present study, we investigated the role of MDSCs in mediating long-term graft survival in almost-tolerant kidney transplant recipients (ATKTRs) and the mechanism(s) responsible for increasing MDSC numbers in these recipients. Methods. Peripheral blood mononuclear cells (PBMCs) from whole blood samples were collected from 30 ATKTRs (graft survival, > 10 years after kidney transplant [KTx]) treated with low doses of immunosuppressive drugs and with stable kidney function, 10 short-term graft survival kidney transplant recipients (STKTRs; graft survival, similar to 1-3 years post-KTx) with stable kidney function, and 10 healthy donors (HDs). MDSC and regulatory T cell (Tregs) levels were analyzed using multicolor flow cytometry in PBMCs. Results. ATKTRs had significantly higher levels of monocytic MDSCs (P < .001) and CD4(+)CD25(+)FoxP3(+) Tregs than STKTRs and HDs. Furthermore, the M-MDSC levels correlated positively with the survival rates, estimated glomerular filtration rates (eGFRs) of grafts, and the levels of CD4(+)CD25(+)FoxP3(+) Tregs in ATKTRs. Conclusions. Accumulation of high levels of MDSCs was observed in ATKTRs. Changes in MDSC levels may play important roles in mediating transplant tolerance and regulating Tregs. Therefore, we propose that MDSCs may be potentially used for recognizing tolerant transplant recipients and guiding dosage reduction for immunosuppressive drugs for KTx.