The maintenance of regulatory T (Treg) cells critically prevents autoimmunity. Pre-B cell leukemia transcription factor 1 (Pbx1) variants are associated with lupus susceptibility, particularly through the expression of a dominant negative isoform Pbx1-d in CD4+ T cells. Pbx1-d overexpression impaired Treg cell homeostasis and promoted inflammatory CD4+ T cells. Here, we showed a high expression of Pbx1 in human and murine Treg cells, which is decreased in lupus patients and mice. Pbx1 deficiency or Pbx1-d overexpression reduced the number, stability, and suppressive activity of Treg cells, which increased murine responses to immunization and autoimmune induction. Mechanistically, Pbx1 deficiency altered the expression of genes implicated in cell cycle and apoptosis in Treg cells. Intriguingly, Rtkn2, a Rho-GTPase previously associated with Treg homeostasis, was directly transactivated by Pbx1. Our results suggest that the maintenance of Treg cell homeostasis and stability by Pbx1 through cell cycle progression prevent the expansion of inflammatory T cells that otherwise exacerbates lupus progression in the hosts.