Pneumolysin (Ply), a major virulence factor of Streptococcus pneumoniae (S. pn), affects the immunity of host cells during infection. It has been reported that Ply is involved in S. pn standard strain D39-induced interferon-β (IFN-β) expression; however, other findings suggest that recombinant Ply protein is incapable of triggering IFN-β expression. Here, we demonstrated that purified Ply was capable of initiating oxidative damage to mitochondria, resulting in the subsequent release of mitochondrial deoxyribonucleic acid (mtDNA), which mediated IFN-β expression in macrophages. Importantly, we determined that IFN-β expression was regulated by stimulator of interferon genes (STING) signaling in response to Ply. In conclusion, our study identified that IFN-β production was triggered by Ply in macrophages and mtDNA released from Ply-damaged mitochondria mediated this process, through the STING pathway. This is a novel mechanism by which S. pn modulates type I IFN response in macrophages.,© 2019 Federation of European Biochemical Societies.