ObjectivesThe mechanism of Brucella infection regulating macrophage phenotype has not been completely elucidated until now. This study aimed to determine the mechanism ofBrucella abortusin the modulation of macrophage phenotype using RAW264.7 cells as a model.Materials and methodsRT-qPCR, ELISA and flow cytometry were used to detect the inflammatory factor production and phenotype conversion associated with M1/M2 polarization of macrophages byBrucella abortusinfection. Western blot and immunofluorescence were used to analyze the role of nuclear factor kappa B (NF-κB) signaling pathway in regulation ofBrucella abortus-induced macrophage polarization. Chromatin immunoprecipitation sequencing (Chip‐seq), bioinformatics analysis and luciferase reporter assay were used to screen and validate NF-κB target genes associated with macrophage polarization and further verify its function.ResultsThe results demonstrate thatB. abortusinduces a macrophage phenotypic switch and inflammatory response in a time-dependent manner.With the increase of infection time,B. abortusinfection-induced M1-type increased first, peaked at 12 h, and then decreased, whereas the M2-type decreased first, trough at 12 h, and then increased. The trend of intracellular survival ofB. abortuswas consistent with that of M2 type. When NF-κB was inhibited, M1-type polarization was inhibited and M2-type was promoted, and the intracellular survival ofB. abortusincreased significantly. Chip‐seq and luciferase reporter assay results showed that NF-κB binds to the glutaminase gene (Gls).Glsexpression was down-regulated when NF-κB was inhibited. Furthermore, whenGlswas inhibited, M1-type polarization was inhibited and M2-type was promoted, the intracellular survival ofB. abortusincreased significantly. Our data further suggest that NF-κB and its key target geneGlsplay an important role in controlling macrophage phenotypic transformation.ConclusionsTaken together, our study demonstrates thatB. abortusinfection can induce dynamic transformation of M1/M2 phenotype in macrophages. Highlighting NF-κB as a central pathway that regulates M1/M2 phenotypic transition. This is the first to elucidate the molecular mechanism ofB. abortusregulation of macrophage phenotype switch and inflammatory response by regulating the key geneGls, which is regulated by the transcription factor NF-κB.