FOXP3+ regulatory T cells (Tregs) are central to the maintenance of self-tolerance and immune homeostasis. The mechanisms of action and cellular targets for Treg-mediated suppression remain controversial. The critical adhesion molecules utilized by Tregs for the interaction with their target cells have not been well characterized. We show that human CD4+ FOXP3+ CD25 high cells (hTregs) suppress the activation of mouse responders as efficiently as mouse Tregs. LFA-1 (CD11a/CD18) on the hTregs is critical for their suppressor function, since suppression can be reversed with blocking anti-hCD11a or anti-hCD18 mAb. Tregs from patients with LFA-1 deficiency fail to suppress human and mouse responders. Mouse CD4+ T cells deficient in ICAM-1 can be suppressed by hTregs, indicating that the hTregs target mouse dendritic cells (DCs) through the binding of human LFA-1 to mouse ICAM-1. Coculture of mouse DCs with hTregs, but not hTregs from LFA-1-deficient patients, prevented the up-regulation of CD80/CD86 on the DCs and their capacity to activate responder T cells. Lastly, IL-2 is not required for hTreg suppressor function under optimal stimulatory condition and IL-2 consumption plays no role in hTreg-mediated suppression. Taken together, one of the mechanisms of Treg-mediated suppression functions across species and mediates an LFA-1/ICAM-1-dependent interaction between Tregs and DCs.