Mastitis is a prevalent disease in dairy cattle that causes large economic loss, mostly through its effects on milk production. Streptococcus agalactiae is a common and major cause of bovine mastitis, but the immune response related to S. agalactiae-induced mastitis is a very complex biological process. To understanding the pathophysiological process and the host immune response to S. agalactiae-induced mastitis, we used nipple tube perfusion of S. agalactiae to establish a Chinese Holstein cattle clinical mastitis model. Visual inspection, somatic cells count analysis, histopathology and transmission electron microscopy examination have confirmed the successful establishment of the S. agalactiae-induced mastitis model. Then, we used Bovine Gene Expression microarray and isobaric tags for relative and absolute quantitation (iTRAQ) to screen potential genes and proteins associated with mastitis. Compared with healthy mammary glands tissue, 129 differentially expressed genes (fold change ≥2, P ≤ 0.05) and 144 differentially expressed proteins (>1.2-fold) were identified. Most significantly, differentially expressed genes were involved in immune and inflammatory reactions, such as complement and coagulation cascades, inflammatory mediator regulation of TRP channels, and bacterial invasion of epithelial cells. iTRAQ analysis also showed that the immune and inflammatory related proteins were significantly upregulated in the mastitis group, such as fibronectin 1, complement factor H, and von Willebrand. In conclusion, this joint analysis of transcriptomic and proteomic profiles provides an enhanced understanding of immune response to S. agalactiae-induced mastitis in bovine mammary glands.