The lethality of infection by Bacillus anthracis is largely due to its plasmid-encoded toxins, which consist of a carrier protein, the protective antigen (PA), in combination with either the lethal-factor or edema-factor moiety. During B. anthracis infections, PA secreted by bacteria binds to membrane receptors of susceptible cells, is cleaved proteolytically, attaches to lethal factor or edema factor, undergoes oligomerization and internalization, and transports its toxin partners to acidic endosomes where they are released into the cytosol. To identify specific host functions that mediate these events, we used RNA encoded by a lentivirus-based library of approximately 40,000 human ESTs to inactivate chromosomal genes in a human cell population, and we isolated clones that survived PA-dependent toxin-induced death. This phenotypic screen and subsequent analysis identified ARAP3, which is a phosphoinositide-binding protein implicated previously in membrane vesicle trafficking and cytoskeletal organization, as a mammalian host-cell gene that is essential for normal anthrax toxicity. ARAP3 deficiency produced by antisense expression of an ARAP3 EST impaired entry of PA and its bound toxigenic moieties into both human and mouse cells, resulting in reduced toxin sensitivity. Our results demonstrate the usefulness of antisense EST libraries for global chromosomal gene inactivation, establish the practicality of loss-of-function phenotypic screens for the identification of genomic loci required for pathogen effects in mammalian cells, and reveal an important role for ARAP3 in cellular internalization of anthrax toxin.