Specialized secretion systems of bacteria evolved for selective advantage, either killing microbial competitors or implementing effector functions during parasitism. Staphylococcus aureus, an invasive pathogen of humans and animals, requires a specialized ESAT-6-like Secretion System (ESS) to secrete proteins (EsxA, EsxB, EsxC, and EsxD) during infection. Here, we identify a novel secreted effector of the ESS pathway, EssD, that functions as a nuclease and cleaves DNA but not RNA. EssI, a protein of the DUF600 family, binds EssD to block its nuclease activity in the staphylococcal cytoplasm. EssD along with EssE, another protein encoded by the staphylococcal ess, forms complexes with other members of the ESS secretion pathway and its substrates, promoting the secretion of EsxA, EsxB, EsxC, EsxD, and EssD. Expression of ess genes is required for S. aureus establishment of persistent abscess lesions following bloodstream infection; however, the mechanisms whereby effectors of the ESS pathway implement their virulence strategies were heretofore not known. An essE knockout mutant, essD knockout mutant, or a variant lacking nuclease activity, essDL546P, elicited a diminished interleukin-12 (IL-12) cytokine response following bloodstream infection of mice, suggesting that the effector function of EssD stimulates immune signaling to support the pathogenesis of S. aureus infections. Thus, essE-mediated secretion of protein effectors, such as EssD, via the ESS pathway may enable S. aureus to manipulate host immune responses by modifying the production of cytokines and eliciting a Th1 polarization.