Bartonella henselae, the agent of cat-scratch disease and vasculoproliferative disorders in humans, is a fastidious facultative intracellular pathogen, whose interaction with macrophages and endothelial cells (ECs) is crucial in the pathogenesis of these diseases. However, little is known about the subcellular compartment in which B. henselae resides. Two hours after infection of murine macrophages and human ECs, the majority of B. henselae-containing vacuoles (BCVs) lack typical endocytic marker proteins, fail to acidify, and do not fuse with lysosomes, suggesting that B. henselae resides in a non-endocytic compartment. In contrast to human umbilical vein endothelial cells, bacterial death and lysosomal fusion with BCVs is apparent in J774A.1 macrophages at 24 h. This phenomenon of delayed lysosomal fusion requires bacterial viability, and is confined to the BCV itself. Using magnetic selection, we enriched for transposon-mutagenized B. henselae trapped in lysosomes of macrophages 2 h after infection. Genes affected appear to be relevant to the intracellular lifestyle in macrophages and ECs and include some previously implicated in Bartonella pathogenicity. We conclude that B. henselae has a specific capacity to actively avoid the host endocytic pathway after entry of macrophages and ECs, from within a specialized non-endocytic membrane-bound vacuole.