Bovine mastitis is an inflammatory response mainly caused by Staphylococcus aureus. Lysin is a cell wall hydrolase encoded and synthesized by a bacteriophage, which can kill specific Gram-positive bacteria. In this study, phage lysin “LysGH15” is used to treat the mice mastitis caused by S. aureus. The purified lysGH15 showed strong bactericidal activity in vitro. When treated with 25μg/mL of the LysGH15, the bacterial counts of S. aureus dropped approximately 5 log units within 10 min. In the in vivo experiments, the administration of LysGH15 significantly (P<0.05) reduced the colonies of S. aureus and alleviated damage to the breast tissue. Also, the levels of IL-6 and TNF-α in breast tissue were significantly decreased. It indicates that the LysGH15 can effectively treat the murine mastitis caused by S. aureus. This study demonstrated the potential of LysGH15 as an alternative to antibiotics for treating bovine mastitis caused by S. aureus