Streptococcus suis serotype 2 (S. suis 2) is an important pathogen, responsible for diverse diseases in swine and human. In this study, we investigated the role of the glutamine synthetase (GlnA) in the pathogenesis of S. suis 2 in mice. To assess the contribution of glutamine synthetase (GlnA) to the virulence of S. suis 2, an knockout mutant (DeltaglnA) unable to produce GlnA was constructed, and the virulence level of wild-type (WT) SC19 and the DeltaglnA mutant strain were compared in an in vitro adherence assay and murine infection models. The data showed that DeltaglnA mutant exhibited a significant decrease in adherence to the epithelial cells HEp-2. The DeltaglnA mutant strain was attenuated and could reduce mortality and morbidity in murine infection models. Furthermore, organ cultures showed that GlnA plays a role in the colonization of the specific organs involved in S. suis infection. Functional complementation of the glnA gene into the knockout mutant DeltaglnA or incubated with extracellular glutamine restored its ability to adhere to the epithelial cells HEp-2. These findings suggested that glnA is required for the full virulence in S. suis 2. Therefore, the DeltaglnA mutant was considered as an attenuated mutant.