CD4⁺CD25⁺Foxp3⁺ regulatory T (Treg) cells can undergo both thymic selection and peripheral expansion in response to self peptides that are agonists for their T cell receptors (TCR). However, the specificity by which these TCR must recognize peptide:MHC complexes to activate Treg cell function is not known. We show that CD4⁺CD25⁺Foxp3⁺ Treg cells can mediate suppression in response to peptides that are only weakly cross‐reactive with the self peptide that induced their formation in vivo. Moreover, suppression could be efficiently activated by peptide analogs that were inefficient at inducing CD69 up‐regulation, and that also induced little or no proliferation of naïve CD4⁺CD25^–Foxp3^– T cells expressing the same TCR. These findings provide evidence that self peptide‐specific CD4⁺CD25⁺Foxp3⁺ Treg cells can exert regulatory function in response to self‐ and/or pathogen‐derived peptides with which they are only weakly cross‐reactive.