Abstract CD4+CD25+ T cells have immunoregulatory and suppressive functions and are responsible for suppressing self-reactive cells and maintaining self-tolerance. In addition to CD4+CD25+ T cells, there is some evidence that a fraction of CD4+CD25− T cells exhibit suppressive activity in vitro or in vivo. We have shown, using aged mice, that aging not only leads to a decline in the ability to mount CD4+CD25− T cell responses, but, at the same time, renders aged CD4+CD25− T cells suppressive. In this study we report two newly established mAbs that could abrogate the suppressive function of aged CD4+CD25− T cells. These mAbs recognized the same protein, the transmembrane phosphatase CD45. Cross-linking of CD45 on aged CD4+CD25− T cells was required for the disruption of their suppressive activity. Surprisingly, these mAbs also abrogated the suppressive action of CD4+CD25+ T cells in vitro. Our results demonstrate an unexpected function of CD45 as a negative regulator neutralizing the suppressive activity of aged CD4+CD25− and young CD4+CD25+ T cells.