Streptococcus constellatus is a member of the Anginosus group streptococci (AGS) and primarily inhabits the human oral cavity. S. constellatus is composed of three subspecies: S. constellatus subsp. constellatus (SCC), S. constellatus subsp. pharyngis and the newly described subspecies S. constellatus subsp. viborgensis. Although previous studies have established that SCC contains β-haemolytic strains, the factor(s) responsible for β-haemolysis in β-haemolytic SCC (β-SCC) has yet to be clarified. Recently, we discovered that a streptolysin S (SLS) homologue is the β-haemolytic factor of β-haemolytic Streptococcus anginosus subsp. anginosus (β-SAA), another member of the AGS. Furthermore, because previous studies have suggested that other AGS species, except for Streptococcus intermedius, do not possess a haemolysin(s) belonging to the family of cholesterol-dependent cytolysins, we hypothesized that, as with β-SAA, the SLS homologue is the β-haemolytic factor of β-SCC, and therefore aimed to investigate and characterize the haemolytic factor of β-SCC in the present study. PCR amplification revealed that all of the tested β-SCC strains were positive for the sagA homologue of SCC (sagA(SCC)). Further investigations using β-SCC strain W277 were conducted to elucidate the relationship between sagA(SCC) and β-haemolysis by constructing sagA(SCC) deletion mutants, which completely lost β-haemolytic activity. This loss of β-haemolytic activity was restored by trans-complementation of sagA(SCC). Furthermore, a co-cultivation assay established that the cytotoxicity of β-SCC was clearly dependent on the presence of sagA(SCC). These results demonstrate that sagA(SCC) is the factor responsible for β-SCC β-haemolysis and cytotoxicity.