Antigen-specific transplantation tolerance in the absence of immunosuppressive drugs is a rarely achieved goal. Immune responses to Y chromosome-encoded transplantation antigens (HY) can have life-threatening consequences in the clinic. Here, we have adopted a procedure developed in T cell antigen receptor (TCR)-transgenic mice to convert naïve T cells into male-specific Foxp3⁺ regulatory T cells (Tregs) in WT female mice. For this purpose, female mice were infused by osmotic minipumps with a single class II MHC-presented HY peptide and Tregs visualized by tetramer staining. As a result, animals developed Treg-mediated long-term tolerance to all HY transplantation antigens, irrespective of whether they were recognized by CD4 or CD8 T cells, on skin or hematopoietic grafts from male donors.