We have previously shown that macrophages (Mφ) can be activated by CD40 ligation to become cytotoxic against tumor cells in vitro. Here we show that treatment of mice with agonistic anti-CD40 mAb (anti-CD40) induced up-regulation of intracellular TLR9 in Mφ and primed them to respond to CpG-containing oligodeoxynucleotides (CpG), resulting in synergistic activation. The synergy between anti-CD40 and CpG was evidenced by increased production of IFN-γ, IL-12, TNF-α, and NO by Mφ, as well as by augmented apoptogenic effects of Mφ against tumor cells in vitro. The activation of cytotoxic Mφ after anti-CD40 plus CpG treatment was dependent on IFN-γ but not TNF-α or NO, and did not require T cells and NK cells. Anti-CD40 and CpG also synergized in vivo in retardation of tumor growth in both immunocompetent and immunodeficient mice. Inactivation of Mφ in SCID/beige mice by silica treatment abrogated the antitumor effect. Taken together, our results show that Mφ can be activated via CD40/TLR9 ligation to kill tumor cells in vitro and inhibit tumor growth in vivo even in immunocompromised tumor-bearing hosts, indicating that this Mφ-based immunotherapeutic strategy may be appropriate for clinical testing.