Coxiella burnetii is the etiological agent of the zoonotic disease Q fever, which is featured by its ability to replicate in acid vacuoles resembling the lysosomal network. One key virulence determinant of C. burnetii is the Dot/Icm system that transfers more than 150 effector proteins into host cells. These effectors function to construct the lysosome-like compartment permissive for bacterial replication, but the functions of most of these effectors remain elusive. In this study, we used an affinity tag purification mass spectrometry (AP-MS) approach to generate a C. burnetii-human protein-protein interaction (PPI) map involving 53 C. burnetii effectors and 3480 host proteins. This PPI map revealed that the C. burnetii effector CBU0425 (designated CirB) interacts with most subunits of the 20S core proteasome. We found that ectopically expressed CirB inhibits hydrolytic activity of the proteasome. In addition, overexpression of CirB in C. burnetii caused dramatic inhibition of proteasome activity in host cells, while knocking down CirB expression alleviated such inhibitory effects. Moreover, we showed that a region of CirB that spans residues 91-120 binds to the proteasome subunit PSMB5 (beta 5). Finally, PSMB5 knockdown promotes C. burnetii virulence, highlighting the importance of proteasome activity modulation during the course of C. burnetii infection.