Polyclonal expansion of human regulatory T cells (Tregs) prevents xenogeneic rejection by suppressing effector T cell responses in vitro and in vivo. However, a major limitation to using polyclonally expanded Tregs is that they may cause pan‑immunosuppressive effects. The present study was conducted to compare the ability of ex vivo expanded human xenoantigen‑stimulated Tregs (Xeno‑Treg) and polyclonal Tregs (Poly‑Treg) to protect islet xenografts from rejection in NOD‑SCID interleukin (IL)‑2 receptor (IL2r)γ‑/‑ mice. Human cluster of differentiation (CD)4+CD25+CD127lo Tregs, expanded either by stimulating with porcine peripheral blood mononuclear cells (PBMCs) or anti‑CD3/CD28 beads, were characterized by immune cell phenotyping, T cell receptor (TCR) Vβ CDR3 spectratyping and performing suppressive activity assays in vitro. The efficiency of adoptively transferred ex vivo human Tregs was evaluated in vivo using neonatal porcine islet cell clusters (NICC) transplanted into NOD‑SCID IL‑2rγ‑/‑ mice, which received human PBMCs with or without Xeno‑Treg or Poly‑Treg. Xeno‑Treg, which expressed increased levels of human leukocyte antigen‑DR and secreted higher levels of IL‑10, demonstrated enhanced suppressive capacity in a pig‑human mixed lymphocyte reaction. Spectratypes of TCR Vβ4, Vβ10, Vβ18 and Vβ20 in Xeno‑Treg showed restriction and expanded clones at sizes of 205, 441, 332 and 196 respectively, compared to those of Poly‑Treg. Reconstitution of mice with human PBMCs and Poly‑Treg resulted in NICC xenograft rejection at 63 days. Adoptive transfer with human PBMCs and Xeno‑Treg prolonged islet xenograft survival beyond 84 days, with grafts containing intact insulin‑secreting cells surrounded by a small number of human CD45+ cells. This study demonstrated that adoptive transfer of ex vivo expanded human Xeno‑Treg may potently prevent islet xenograft rejection in humanized NOD‑SCID IL2rγ‑/‑ mice compared with Poly‑Treg. These findings suggested that adoptive Treg therapy may be used for immunomodulation in islet xenotransplantation by minimizing systemic immunosuppression.