Virulent strains of Histophilus somni are important pathogens of cattle, are recognized to survive within phagocytic cells, and are serum-resistant. The fic motif within the IbpA surface protein is toxic for host cells, but it is not known whether persistence within phagocytic cells is due to toxicity. Disease and commensal isolates, and strains with mutations within ibpA were used with a macrophage cell line (BM) to follow intracellular survival and trafficking to determine the mechanism of H. somni intracellular survival. Intracellular bacterial trafficking was determined using confocal microscopy and Alexa Fluor 488 or 546-labelled monoclonal antibodies to early phagosome and late lysosomal markers. Phagosome acidification was determined using Lysotracker. H. somni strains expressing IbpA, or strains with mutations in ibpA outside the fic motif, were serum-resistant. However, mutants with all of IbpA, or only the fic motif, deleted became serum-sensitive. Incubation of H. somni strain 2336 with BM cells caused the cells to round up, but the bacteria survived within these cells for at least 72 h. In contrast, commensal strain 129Pt, lacking IbpA, was not toxic for macrophages and did not survive 24 h within BM cells. Strain 2336 with transposon and allelic exchange mutations in ibpA outside the fic motif remained toxic for BM cells and survived intracellularly. However, mutants with the entire ibpA gene, or only the fic motif, deleted were not toxic for BM cells, but the mutants survived intracellularly as well as the parent. Early phagosomal marker EEA-1 co-localized with both strains 2336 and 129Pt. However, strain 2336 and other virulent strains that survived intracellularly prevented acidification of phagosomes and did not co-localize with late lysosomal marker LAMP-2 (P<0.05). These results suggest that intracellular survival was, in part, due to prevention of phagosome-lysosome fusion, and that virulent H. somni strains may be permissive intracellular pathogens.