Cytotoxic T lymphocyte (CTL) responses are initiated by dendritic cells (DC) and are required for immune-mediated clearance of tumours and many pathogens for which there are currently no effective vaccines. Targeting antigen (Ag) to DC in vivo is an attractive strategy for vaccine development and clinical trials are currently underway using antibodies (Ab) specific for the DEC-205 receptor to deliver tumour Ag to DC in patients with solid malignancies. However, DEC-205 is widely expressed on all human DC subsets and other cell types, which may compromise targeting efficiency. Human CD141+ DC have been identified as the main subtype involved in Ag cross-presentation and are considered the most effective subset to target for CTL induction. The C-type lectin-like receptor CLEC9A is specifically expressed on CD141+ DC, binds actin filaments exposed by dead cells and facilitates cross-presentation of dead cell Ag, making it an attractive candidate for specifically targeting this DC subset. We have produced and validated recombinant human chimaeric IgG4 Ab specific for human CLEC9A, DEC-205 and an isotype control. We fused a fragment of the human CMV pp65 Ag, including HLA-A2 and HLA-DR3 restricted epitopes to the Ab heavy chains to create Ab-Ag fusion proteins. These fusion proteins retain binding specificity for their target receptor, are internalised and accumulate at equivalent rates by CD141+DCs. Following uptake of DEC-205-pp65, both CD141+ DC and CD1c+ DC presented pp65 to Ag-specific CD4+ T cells but failed to cross-present the HLA-A2-restricted epitope to Ag-specific CD8+ T cells. In contrast Clec9A-pp65 Ab specifically delivered pp65 to CD141+ DCs for presentation to both CD8+ and CD4+ pp65-specific T cells. Thus, targeting of human CD141+ DC via CLEC9A results in superior cross-presentation to CD8+ T cells and similar presentation to CD4+ T cells compared to DEC-205 and warrants development as an immunotherapeutic for cancer or infectious disease.