Brucella spp., an intracellular bacterium, uses its type Ⅳ secretion system (T4SS) to regulate host signaling pathways and promote intracellular survival, but the molecular mechanism of this process remains largely unknown. Here we found that increasing the abundance of acetylated protein in host cells promotes the intracellular survival of Brucella. Moreover, our results demonstrated that the Brucella effector protein BspF can impact protein acetylation modification in host cells by interacting with other intracellular acetyltransferases. We conducted liquid chromatography-tandem mass spectrometry (LC-MS/MS) to characterize the protein acetylation mediated by BspF. We identified that synaptosome associated protein 29 (SNAP29) K103 was acetylated, and that acetylated SNAP29 inhibited its interaction with syntaxin 17 (STX17), thereby regulating the autophagy and providing an environment for the intracellular survival of Brucella. Furthermore, our results provide the first report of a bacterial effector using acetylation to affect the SNAP29-STX17-VAMP8 complex, and inhibit the host’s defense system. Our results suggest a vital role of SNAP29 acetylation in autophagy of host cells under intracellular infection, by specifically regulating the assembly of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE).