Simple SummaryStreptococcus suishas been receiving increasing attention due to its involvement in severe human infections worldwide. However, there is currently no licensed vaccine againstS. suis. Therefore, vaccine development is very essential to eliminate the threat of this human pathogen. Herein, we contrived a multi-epitope subunit vaccine againstS. suisisolates of multiple serotypes using immunoinformatics. Further, the characteristics of the designed vaccine were evaluated using several analyses, including antigenicity, allergenicity, toxicity, and tertiary structure analyses and molecular docking; the results indicated that it could be an effective universal vaccine againstS. suisinfections.AbstractStreptococcus suisis a significant zoonotic pathogen that is a great threat not only to the swine industry but also to human health, causing arthritis, meningitis, and even streptococcal toxic shock-like syndrome. Owing to its many serotypes and high geographic variability, an efficacious cross-protectiveS. suisvaccine is not readily available. Therefore, this study aimed to design a universal multi-epitope vaccine (MVHP6) that involved three highly immunogenic proteins ofS. suis, namely, the surface antigen containing a glycosaminoglycan binding domain (HP0197), endopeptidase (PepO), and 6-phosphogluconate dehydrogenase (6PGD). Forecasted T-cell and B-cell epitopes with high antigenic properties and a suitable adjuvant were linked to construct a multi-epitope vaccine. In silico analysis showed that the selected epitopes were conserved in highly susceptible serotypes for humans. Thereafter, we evaluated the different parameters of MVHP6 and showed that MVHP6 was highly antigenic, non-toxic, and non-allergenic. To verify whether the vaccine could display appropriate epitopes and maintain high stability, the MVHP6 tertiary structure was modeled, refined, and validated. Molecular docking studies revealed a strong binding interaction between the vaccine and the toll-like receptor (TLR4), whereas molecular dynamics simulations demonstrated the vaccine’s compatibility, binding stability, and structural compactness. Moreover, the in silico analysis showed that MVHP6 could evoke strong immune responses and enable worldwide population coverage. Moreover, MVHP6 was cloned into the pET28a (+) vector in silico to ensure the credibility, validation, and proper expression of the vaccine construct. The findings suggested that the proposed multi-epitope vaccine can provide cross-protection againstS. suisinfections.