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.