Updated publication reference for PubMed record(s): 26873878, 30161230. Previous studies indicated that B. pertussis, historically considered as extracellular pathogen, can invade and replicate within human macrophages. In order to investigate factors involved in the intracellular survival, we analyzed the proteome of intracellular B. pertussis at 3h and 48 h post macrophage infection. From totally 762 identified proteins, we observed differences in gene expression profiles of intracellular bacteria as compared to extracellular bacteria. More than 300 proteins showed significant differences in the level of expression (p< 0.05) between internalized and non-internalized samples. Different intensities were found for proteins associated with virulence, stress response, cell division, transport, and many genes with unknown functions. Proteins involved in stress response increased in level early after infection, including ClpX, GroEL and HsIU. Two proteins involved in iron uptake were found increased as early as 3 h post infection and remained at high level 48 h post-infection. These proteins are BfrD and BfrE, recently identified as TonB-dependent outer membrane catecholamine receptors involved in iron uptake from transferrin in B. bronchiseptica. Interestingly, we found at least three proteins of the Type III Secretion System (TTSS) increased already at 3 h after infection, the proteins BteA, BscC, and BopN suggesting that TTSS becomes operative when bacteria infect human macrophages. Selected virulence factors and metabolic enzymes were validated using the multiple reaction monitoring. These results represent the first characterization of intracellular B. pertussis proteome and demonstrate that this pathogen undergoes an adaptive response that enables the development of an intracellular infection.