Bartonellaspecies are Gram-negative, emerging bacterial pathogens found in two distinct environments. In the gut of the obligately hematophagous arthropod vector, bartonellae are exposed to concentrations of heme that are toxic to other bacteria. In the bloodstream of the mammalian host, access to heme and iron is severely restricted. Bartonellae have unusually high requirements for heme, which is their only utilizable source of iron. Although heme is essential forBartonellasurvival, little is known about genes involved in heme acquisition and detoxification. We developed a strategy for high-efficiency transposon mutagenesis to screen for genes inB. henselaeheme binding and uptake pathways. We identified aB. henselaetransposon mutant that constitutively expresses the hemin binding protein C (hbpC) gene. In the wild-type strain, transcription ofB. henselae hbpCwas upregulated at arthropod temperature (28°C), compared to mammalian temperature (37°C). In the mutant strain, temperature-dependent regulation was absent. We demonstrated that HbpC binds hemin and localizes to theB. henselaeouter membrane and outer membrane vesicles. Overexpression ofhbpCinB. henselaeincreased resistance to heme toxicity, implicating HbpC in protection ofB. henselaefrom the toxic levels of heme present in the gut of the arthropod vector. Experimental inoculation of cats withB. henselaestrains demonstrated that both constitutive expression and deletion ofhbpCaffect the ability ofB. henselaeto infect the cat host. Modulation ofhbpCexpression appears to be a strategy employed byB. henselaeto survive in the arthropod vector and the mammalian host.