The Gram-positive bacterium Staphylococcus aureus is an opportunistic human pathogen that causes a wide spectrum of acute and chronic infections. Its genome encodes two thermonuclease-like homologs nuc1 and nuc2. The mature form of Nuc1, known as NucA, is secreted and plays an important role in virulence. NucA degrades extracellular DNA (eDNA) as well as RNA, and contributes to bacterial escape from neutrophil extracellular traps (NET) and killing, thereby boosting staphylococcal persistence. Besides, it also disrupts e-DNA based biofilm matrix and contributes to bacterial dispersion and spreading. Here we show that uncut bacterial DNA induces production of TNF-α by murine macrophages (RAW264.7), however, pretreatment of DNA with NucA abrogated this activity. Live S. aureus JE2 and JE2Δnuc1 similarly immuno-stimulated RAW264.7, MM6 monocytes, HaCaT keratinocytes, and SAOS-2 osteoblast-like cells. However, JE2Δnuc1 showed decreased host cell invasion, was killed much better than the parent strain by human primary neutrophils and induced less cytokines (TNF-α and IL10). In a septic arthritis mouse model the pathogenicity of Δnuc1 was largely attenuated as compared to the parental S. aureus Newman strain. Δnuc1-infected mice showed almost no weight loss, mild symptoms of clinical arthritis, largely decreased kidney abscess score and bone destruction, and produced less cytokines. The results suggest that Nuc1 is an important virulence factor during S. aureus infection.