Objective To study the effect of daphnetin（DAPH） on the antibacterial activity of macrophages. Methods Firstly， the safety concentration of DAPH（in a range of 10， 20， 40， 80 and 160μM） on macrophages was determined by MTT. Then RAW264. 7 cell lines or murine peritoneal macrophages were pretreated with DMSO and DAPH， followed by S. aureus infection. The phagocytic activity was analyzed by flow cytometry and the bactericidal capability was tested by CFU counting method in DAPH-treated macrophages. Also， the expression of inflammatory cytokines（IL-6， TNF-α and IL-1β） and antimicrobial effector molecules（IL-22， Reg3β， S100A8 and Miz1） were assayed by quantitative PCR. Results Daphnetin treatment significantly enhanced the bacteria-clearance capability of macrophages， although it did not affect cellular phagocytosis. Associated with this， daphnetin remarkably increased the capability of macrophages to express antimicrobial effector molecules while repressing their production of inflammatory cytokines. Conclusion Daphnetin has a protective effect on S. aureus infection with both anti-bacterial and anti-inflammatory properties.