Streptococcus equi ssp. zooepidemicus (SEZ) is responsible for a wide variety of infections in many species, including pigs, horses and humans. Biofilm formation is essential for pathogenesis, and the ability to resist antibiotic treatment results in difficult-to-treat and persistent infections. However, the ability of SEZ to form biofilms is unclear. Furthermore, the mechanisms underlying SEZ biofilm formation and their attributes are poorly understood. In this study, scanning electron microscopy (SEM) demonstrated that SEZ strain ATCC35246 formed biofilms comprising a thick, heterogeneous layer with clumps on the coverslips when incubated for 24 h. In addition, we used a two-dimensional gel electrophoresis (2-DE) based approach to characterize differentially expressed protein in SEZ biofilms compared with their planktonic counterparts. The results revealed the existence of 24 protein spots of varying intensities, 13 of which were upregulated and 11 were downregulated in the SEZ biofilm compared with the planktonic controls. Most of proteins expressed during biofilm formation were associated with metabolism, adhesion, and stress conditions. These observations contribute to our understanding of the SEZ biofilm lifestyle, which may lead to more effective measures to control persistent SEZ infections.