Mastitis in dairy cattle causes significant economic losses through the decreased production and quality of milk. Pathogenic microbial infection is the main cause of mastitis, with Staphylococcus aureus being a major causative pathogen. Staphylococcus aureus can colonize and multiply in the mammary gland, and can enter and persist in cells. Most antibiotics cannot enter cells or may be inefficient if their cellular concentration is too low. As with many bacteria, S. aureus can develop resistance to the antibiotic being used. Residual antibiotics in milk present significant food safety problems for human health. Therefore, non-antibiotic methods for the prevention and treatment of bovine mastitis are of great interest. We used teat tube perfusion to establish a Chinese Holstein cattle clinical mastitis model. Clinical examination, Beijing mastitis test, microscopy enumeration of somatic cell count, and histopathology confirmed the successful establishment of the mastitis model. The influence of mastitis on the mammary tissue proteomic profile was investigated using isobaric tags for relative and absolute quantitation. Healthy and diseased mammary tissue were screened and differentially expressed proteins were identified. Bioinformatic analysis identified proteins related to inflammation and immunization of dairy cows. In this study, the immune response of dairy cow to S. aureus was explored. Western blotting and immunohistochemical staining were used to study the levels of PGLTRP1 and PTX3 proteins in mammary gland tissue and to characterize the changes of these immune-related candidate proteins. The results show that PRLYRP1 and PTX3 play an important role in the development of bovine mastitis and future studies of PRLYRP1 and PTX3 are warranted.