Infection with Mycoplasma hyopneumoniae (M. hyopneumoniae) leads to chronic infectious pneumonia in pigs, resulting in significant distress and economic losses in the global pig industry. The pathogen secretes various proteins, including toxins, adhesins, and virulence-related enzymes, which facilitate adhesion, invasion, and immune evasion processes between bacteria and the host. However, the effector proteins of M. hyopneumoniae are predominantly uncharacterized. In this study, we demonstrate that the nuclease Mhp597 functions as a potential effector protein of M. hyopneumoniae, and we elucidate its mechanism of action in facilitating immune evasion. Our findings indicate that Mhp597 exhibits high expression efficiency in host cells and significantly inhibits IFN-α and IFN-β protein expression. Using yeast two-hybrid and co-immunoprecipitation experiments, we established that Mhp597 interacts with porcine alveolar macrophage vimentin (Vim) via specific amino acid residues (Arg 232, Lys 256, Phe 263, and Lys 317). Further analysis revealed that Mhp597 inhibited the phosphorylation of TBK1 and IRF3 via Vim, thereby suppressing type I interferon (IFN-I) production and promoting the proliferation of M. hyopneumoniae within host cells. In conclusion, this study provides the first detailed account of the molecular mechanism by which Mhp597 negatively regulates the TBK1-IRF3-IFN-I signaling pathway through Vim, thus facilitating immune evasion and proliferation of M. hyopneumoniae within host cells. These findings enhance our understanding of the pathogenic mechanisms of M. hyopneumoniae and suggest potential molecular targets for the development of novel therapeutic strategies.