Streptococcus suis is a commensal zoonotic pathogen contributing to high mortality rates and significant economic loses within the pork production industry. Zoonotic transmission to humans is associated with severe diseases, which poses a global health risk. The diverse bacterial capsular polysaccharide (CPS) between different S. suis serotypes remains an obstacle in vaccine development. Prevention of widespread S. suis infections rely heavily on autogenous vaccines, which offer limited strain-specific responses. With no commercial serotype-independent vaccines available, the present study aimed to develop a novel gamma-irradiated whole-cell S. suis vaccine (Gamma-SU) and to test cross-protective responses. The vaccine strain was genetically modified by deleting the cpsF gene involved in CPS biosynthesis to help attenuation and exposure of surface protein antigens. In addition, the suilysin toxin was detoxified to reduce virulence and enhance vaccine safety. We then assessed the inactivation kinetics upon exposure to gamma-irradiation and defined a sterilising dose for vaccine generation. Integrity of Gamma-SU relative to live bacteria was assessed through scanning and transmission electron microscopy, complemented by assessing the protein profile and vaccine stability using SDS-PAGE. The ability of Gamma-SU to activate Toll-like receptor 2 (TLR2) was assessed in vitro and the immunogenicity was explored in a mouse model. Our data illustrate the structural integrity of Gamma-SU and the induction of positive immune responses. Overall, we have successfully developed a novel vaccine against S. suis with the potential to elicit serotype-independent protection, making the vaccine suitable for further development. If effective, this vaccine has the potential to benefit the pork production industry and offer a vaccine to help prevent zoonotic infections in humans.