CpG oligonucleotides are short single-stranded synthetic DNA molecules. Upon binding to Toll-like receptor 9 (TLR9), CpG activates immune cells in humans and mice. This results in robust Th1 type immunity potentially resulting in clearance of pathogens, reduction of allergy and anti-tumor immunity. However, the effectiveness of CpG as an adjuvant depends on its administration route, with only strong effects seen when CpG is administered locally. As local administration is not always feasible, we generated conjugates to specifically deliver CpG to myeloid cells often abundantly present in tumors. For this we coupled CpG (3′-Thiol-modified phosphorothioate (PTO) CpG-ODN1826 type B (5′-tccatgacgttcctgacgtt-3′)) to monoclonal antibodies (mAbs) directed against the myeloid cell marker CD11b using maleimide-thiol coupling. The CD11b-CpG mAb (αCD11b-CpG) conjugates contained about four CpG molecules/conjugate and displayed binding and internalization characteristics similar to unconjugated CD11b mAbs (αCD11b). The αCD11b-CpG conjugates readily induced maturation of murine dendritic cells (DCs) in a TLR9-dependent manner in vitro. Following intravenous injection, αCD11b-CpG conjugates efficiently targeted CD11b+ immune cells in the blood, lymph nodes and spleen. Finally, injection of αCD11b-CpG conjugates, but not untargeted conjugates, induced maturation of CD11b+ cell subsets in vivo. In conclusion, conjugating CpG to αCD11b enabled specific targeting and activation of myeloid cells in vivo. Display omitted •CpG oligonucleotides can be efficiently coupled to anti-CD11b antibodies.•Anti-CD11b-CpG antibody conjugates are readily internalized by CD11b+ myeloid cells.•Delivery of CpG via antibody-conjugates potently activates myeloid cells via TLR9.•Anti-CD11b-CpG conjugates target and activate CD11b+ myeloid cell subsets in vivo.