Abstract CD3 specific monoclonal antibody therapy uses multiple mechanisms to induce immune tolerance. In this report, we determined that intra-peritoneal administration of 10 µg of mitogenic anti-CD3 monoclonal antibody (mAb) into C57BL/6 mice resulted in a significant reduction in the frequency of splenic dendritic cells (CD11chi) when compared to PBS treated mice at 24h post-injection. Interestingly, the proportion of CD11chi cells undergoing apoptosis (as determined by Apo-stat assay) was significantly higher in anti-CD3 treated mice in comparison to PBS treated control group. Recently, it was shown that in vitro activation of CD4+CD25+ regulatory T cells with anti-CD3 mAb induces granzyme B expression in regulatory T cells and transforms them into cytolytic T cells. To determine the underlying mechanism of increased apoptosis of dendritic cells in anti-CD3 mAb treated mice, we next ascertained the expression of granzyme B in CD4+Foxp3+ regulatory T cells (Treg) derived from the spleen tissue of anti-CD3 and PBS treated groups of mice. Our results showed that when restimulated in vitro with anti-CD3 mAb (1 µg/ml) for 6h, frequency of Treg expressing cell surface CD107a and intracellular granzyme B was significantly higher in anti-CD3 treated mice in comparison to PBS treated control group. Taken together, our results determined that in vivo administration of anti-CD3 mAb resulted in the induction of granzyme B expressing Treg pool and enhanced apoptosis of dendritic cells.