Toxin-antitoxin (TA) systems are widely prevalent in the genomes of bacteria and archaea. These modules have been identified in Escherichia coli and various other bacteria. However, their presence in the genome of Streptococcus suis, an important zoonotic pathogen, has received little attention. In this study, we describe the identification and characterization of a type II TA system, comprising the chromosomal yefM-yoeB locus of S. suis. The yefM-yoeB locus is present in the genome of most serotypes of S. suis. Overproduction of S. suis YoeB toxin inhibited the growth of E. coli, and the toxicity of S. suis YoeB could be alleviated by the antitoxin YefM from S. suis and Streptococcus pneumoniae, but not by E. coli YefM. More importantly, introduction of the S. suis yefM-yoeB system into E. coli could affect cell growth. In a murine infection model, deletion of the yefM-yoeB locus had no effect on the virulence of S. suis serotype 2. Collectively, our data suggested that the yefM-yoeB locus of S. suis is an active TA system without the involvement of virulence.