Objective To review the characteristics of regulatory T cells (Tregs) and ex vivo expansion of Tregs for treatment of graft-versus-host disease (GVHD). Data sources The data used in this review were retrieved from PubMed (1970-2013). The terms "ex vivo expansion", "regulatory T cell", and "graft-versus-host disease" were used for literature search. Study selection The publications about the characteristics of Tregs, ex vivo expansion of Tregs and clinical applications of Tregs against GVHD were identified, retrieved and reviewed. Results Tregs can be classified as natural Tregs (nTregs) and induced Tregs (iTregs). Both subsets share most Treg features. Given their immunosuppressive property, Tregs have been tested for their capability of preventing GVHD. The bottleneck of Treg therapy is the limited numbers of naturally existing Tregs. To solve this problem, ex vivo expansion of nTregs or iTregs has been executed. The initial data indicate Treg therapy is effective in reducing GVHD without compromising graft-versus-leukemia (GVL). Conclusion Ex vivo expansion of Tregs is a reliable way to prepare sufficient number of Tregs for management of GVHD.