Mastitis is a highly prevalent disease in dairy cows that causes large economic losses. Streptococcus agalactiae is a common contagious pathogen and a major cause of bovine mastitis. The immune response to intramammary infection with S. agalactiae in dairy cows is a very complex biological process. To understand the host immune response to S. agalactiae-induced mastitis, mammary gland of lactating Chinese Holstein cows was challenged with S. agalactiae via nipple tube perfusion. Visual inspection, analysis of milk somatic cell counts, histopathology, and transmission electron microscopy of mammary tissue were performed to confirm S. agalactiae-induced mastitis. Microarray and isobaric tags for relative and absolute quantitation (iTRAQ) were used to compare the transcriptomes and proteomes of healthy and mastitic mammary tissue. Compared with healthy tissue, a total of 129 differentially expressed genes (DEGs, fold change >2, p < 0.05) and 144 differentially expressed proteins (DEPs, fold change >1.2, p < 0.05) were identified in mammary tissue from S. agalactiae-challenged cows. Among the concordant 18 DEGs/DEPs, immunoglobulin M precursor, cathelicidin-7 precursor, integrin alpha-5, and complement C4-A-like isoform X1 were associated with mastitis. Intramammary infection with S. agalactiae triggered a complex host innate immune response that involved complement and coagulation cascades, ECM-receptor interaction, focal adhesion, and phagosome and bacterial invasion of epithelial cells pathways. These results provide candidate genes or proteins for further studies in the context of prevention and targeted treatment of bovine mastitis.