IL‐10‐producing CD4⁺ type 1 regulatory T (Tr1) cells, defined based on their ability to produce high levels of IL‐10 in the absence of IL‐4, are major players in the induction and maintenance of peripheral tolerance. Tr1 cells inhibit T‐cell responses mainly via cytokine‐dependent mechanisms. The cellular and molecular mechanisms underlying the suppression of APC by Tr1 cells are still not completely elucidated. Here, we defined that Tr1 cells specifically lyse myeloid APC through a granzyme B (GZB)‐ and perforin (PRF)‐dependent mechanism that requires HLA class I recognition, CD54/lymphocyte function‐associated antigen (LFA)‐1 adhesion, and activation via killer cell Ig‐like receptors (KIRs) and CD2. Notably, interaction between CD226 on Tr1 cells and their ligands on myeloid cells, leading to Tr1‐cell activation, is necessary for defining Tr1‐cell target specificity. We also showed that high frequency of GZB‐expressing CD4⁺ T cells is detected in tolerant patients and correlates with elevated occurrence of IL‐10‐producing CD4⁺ T cells. In conclusion, the modulatory activities of Tr1 cells are not only due to suppressive cytokines but also to specific cell‐to‐cell interactions that lead to selective killing of myeloid cells and possibly bystander suppression.