Chimeric antigen receptor (CAR) technology has propelled T cell engineering. CAR T cells now represent the most promising strategy to treat several cancers. CD19 CARs with either a CD28 or a 4-1BB cytoplasmic domain fused to CD3z have been approved in the clinic. Regulatory T cells (Tregs) offer great promise as next-generation adoptive cell therapies for autoimmune disorders, transplant rejection, and graft-versus-host disease (GvHD). Yet, utilizing CARs to redirect Tregs remains largely unexplored. Here, we generated CD19 CAR Tregs featuring different signaling domains. 28z and 4-1BBz CAR Tregs expressed high levels of Treg markers, did not secrete IL-2, displayed low levels of Treg-specific demethylated region (TSDR) methylation, and suppressed T cell proliferation in vitro. Unexpectedly, CAR-mediated Treg activation also induced expression of perforin and granzyme B, as well as inflammatory cytokines, namely IFN-g and TNF-a. When coadministered with CD19-expressing leukemia cells and bulk T cells into NSG mice, CD19 CAR Tregs suppressed T cell proliferation and prevented GvHD. Surprisingly, however, CAR Tregs also controlled CD19-expressing tumor growth for several weeks with potencies comparable to bulk CAR T cells. Bulk T cell or recombinant IL-2 infusion did not prevent CAR Treg-mediated tumor control. Yet, this phenomenon was dependent on CAR costimulation, as z CAR Tregs failed to control tumor growth in the presence of bulk T cells. Altogether, these data suggest that CAR activation can activate a cytotoxic program in Tregs, making them effective hematological tumor killers in vivo. Experiments dissecting the mechanism of CAR Treg-mediated tumor killing are ongoing.