Streptococcus agalactiae is one of the primary pathogens responsible for subclinical mastitis, a significant economic burden for dairy farms. An essential component of the immune response to infection is ubiquitination, which plays important roles in the complex interactions between the pathogen and host. In the present study, quantitative ubiquitylomics was performed to profile changes in the global ubiquitinome of bovine mammary gland epithelial cells (BMECs) infected with S. agalactiae. The most notable changes in the BMEC ubiquitinome were related to the adherens junction, ribosome, and tight junction pathways. Ubiquitination of CTNNB1, EGFR, ITGB1, CTNNA1, CTNNA2, CDH1, YES1, and SLC9A3R1 appears to be fundamental for regulating multiple cellular processes in BMECs in response to S. agalactiae infection. In addition, broad ubiquitination of various effectors and outer membrane proteins was observed. Ubiquitinated proteins in S. agalactiae-infected BMECs were associated with regulating cell junctions in the host, with potential implications for susceptibility to infection. The preliminary findings suggest that extensive ubiquitination of CTNNB1, CDH1 and SLC9A3R1 and proteins closely related to cell junctions might play an important role in mastitis progression in dairy cows. The results provide evidence that ubiquitin modification of certain proteins in S. agalactiae-infected BMECs could be a promising therapeutic strategy for reducing mammary gland injury and mastitis.