Streptococcus agalactiae is one of the causative agents of subclinical mastitis, a common disease of dairy cows that causes great economic losses in the industry worldwide. It is thought that pathology is mainly due to infammatory damage of bovine mammary epithelial cells (bMECs); however, the mechanism by which S. agalactiae damages the bMECs is not clear. The aim of this study was to evaluate the infammatory efects of S. agalactiae on bMECs and the resulting changes in protein profles. The bMECs were incubated with S. agalactiae for diferent times and assayed for cell viability by MTT assay, apoptosis by annexin V and propidium iodide dual staining, and morphological and ultrastructural changes by scanning and transmission electron microscopy. Quantitative real-time PCR was used to determine the efect of S. agalactiae on expression of mRNA of infammatory factors in bMECs and protein levels were quantitated by liquid chromatography/mass spectrometry. Exposure to S. agalactiae signifcantly decreased the cell viability and triggered apoptosis, as well as up-regulating TNF-α, IL-1β and IL-6 mRNA, and inhibiting IL-8 expression. S. agalactiae also induced morphological and ultrastructural changes. Furthermore, we identifed 325 up-regulated and 704 down-regulated proteins in the treated vs control group. All signifcant diferentially expressed proteins (DSEPs) were classifed into three major areas by function: biological processes, cellular components and molecular functions. These diferentially expressed proteins included enzymes and proteins associated with various metabolic processes and cellular immunity. Pathway enrichment analysis showed that eight down-regulated signaling pathways were signifcantly enriched. Exposure to even subclinical levels of S. agalactiae can lead to infammation and bMEC damage. Our data suggest some possible molecular mechanisms for the harmful efects of subclinical mastitis in dairy cows.