Plasmacytoid dendritic cells (pDCs) are a subset of DCs generally associated with poor prognosis of cancer patients. However, upon TLR7/8 activation by imiquimod, pDCs have been shown to acquire tumor-killing abilities. In this study, by performing multi-omics profiling of imiquimod-stimulated murine bone marrow-derived pDCs (BM-pDCs), we identified MAPKs, JAK/STAT, and NF-κB pathways as critical mediators of the killing function. Analysis of secreted and surface markers in perturbed BM-pDCs revealed that a complex signaling network is necessary to shape the cytotoxic phenotype of imiquimod-activated BM-pDCs. While JNK inhibition reduces killing via secreted factors, p38 inhibition enhances cell-mediated killing. Instead, NF-κB inhibition boosts whereas Tyk2 deficiency reduces both cell- and secreted factors-mediated killing. Data integration identified a pDC killing gene signature with prognostic value in melanoma patients. These results will be instrumental to design further functional studies and clinical approaches to harness the anti-tumorigenic functions of pDCs, in turn improving cancer patients' outcome.