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According to the whole‐genome bioinformatics analysis, a heme‐binding protein from Nocardia seriolae (HBP) was found. HBP was predicted to be a bacterial secretory protein, located at mitochondrial membrane in eukaryotic cells and have a similar protein structure with the heme‐binding protein of Mycobacterium tuberculosis, Rv0203. In this study, HBP was found to be a secretory protein and co‐localized with mitochondria in FHM cells. Quantitative analysis of mitochondrial membrane potential value, caspase‐3 activity, and transcription level of apoptosis‐related genes suggested that overexpression of HBP protein can induce cell apoptosis. In conclusion, HBP was a secretory protein which may target to mitochondria and involve in cell apoptosis in host cells. This research will promote the function study of HBP and deepen the comprehension of the virulence factors and pathogenic mechanisms of N. seriolae.