Type IV secretion systems (T4SSs) are multicomponent machineries that play an essential role in pathogenicity of many facultative intracellular bacteria. The virB operon of Brucella abortus codes for a T4SS essential for virulence and intracellular multiplication. Here, virB expression analyses carried out using lacZ transcriptional fusions showed that virB promoter (PvirB) is temporally activated within J774 cells. Primer extension experiments revealed that virB transcription starts at 27 bp upstream of the first gene of the virB operon. Structural analyses showed that PvirB and regulatory sequences involved in intracellular regulation span 430 bp upstream of the transcription start site. A protein able to bind PvirB was isolated and identified. This protein, homologue to integration host factor (IHF), specifically interacts with PvirB and induces a DNA bending with an angle of 50.36°. DNAse I footprinting experiments showed that IHF protects a 51 bp region that contains two overlapped IHF binding consensus motifs. VirB expression experiments carried out with PvirB-lacZ fusions showed that in B. abortus IHF participates in the regulation of PvirB activity during the intracellular and vegetative growth in different media. A mutant strain with a 20 bp IHF binding site replacement failed to turn on the virB operon during the initial stages of macrophage infection and displayed severe intracellular multiplication defects. These data indicate that IHF plays a key role during intracellular virB operon expression being required for the biogenesis of the endoplasmic reticulum-derived replicative vacuole.