Caspases are classified as inflammatory or apoptotic category. Inflammatory caspases participate in inflammasome activation, while apoptotic caspases mediate apoptotic activation. Previous studies have shown that apoptotic caspases prevent the production of IFN-β during apoptosis or virus infection. However, the relationship between apoptotic caspases and IFN-β production during intracellular bacterial infection is still unclear. Here, we investigated the role of apoptotic caspases in IFN-β production induced by Mycobacterium bovis (M. bovis) infection. M. bovis is an intracellular bacterium and belongs to the Mycobacterium tuberculosis complex. M. bovis infection can cause tuberculosis in animals and human beings. In the current study, we found that M. bovis infection triggered mitochondrial stress, which caused the leakage of cytochrome c into the cytoplasm, and in turn, activated the downstream caspase-9 and-3. Furthermore, our results showed that activation of apoptotic caspases reduced IFN-β production during M. bovis infection and vice versa. Confocal microscopy analysis revealed that apoptotic caspases prevented IFN-β production by decreasing p-IRF3 nuclear translocation. Our findings demonstrate that apoptotic caspases negatively regulate the production of IFN-β induced by an intracellular bacterial infection.