Dendritic cells (DCs) are well-known for their functions in orchestrating the innate and adaptive arms of immune defense. However, under certain conditions, DCs can exert tumoricidal activity. We have elucidated the mechanism of tumor suppression by TLR4-activated bone marrow-derived DCs (BMDCs) isolated from BALB/c mice. We identified that two distinct subsets of BMDCs (CD11b⁺CD11c⁺I-A/E^int and CD11b⁺CD11c⁺I-A/E^high) have different cytotoxic mechanisms of action. The cytotoxicity of the former subset is mediated through NO and reactive oxygen species and type I IFN (IFN-β), whereas the latter subset acts only through IFN-β. TLR4 agonists, LPS or pharmaceutical-grade ImmunoMax, activate CD11c⁺ BMDCs, which, in turn, directly kill 4T1 mouse breast cancer cells or inhibit their proliferation in an MHC-independent manner. These data define two populations of BMDCs with different mechanisms of direct cytotoxicity, as well as suggest that the I-A/E^int subset could be less susceptible to counteracting mechanisms in the tumor microenvironment and support investigation of similar subsets in human DCs.