Few studies have been able to provide experimental evidence of the ability of fleas to maintain rodent-associated Bartonella infections and excrete these bacteria.These data are important for understanding the transmission cycles and prevalence of these bacteria in hosts and vectors. We used an artificial feeding approach to expose groups of the oriental rat flea (Xenopsylla cheopis Rothschild; Siphonaptera, Pulicidae) to rat blood inoculated with varying concentrations of Bartonella elizabethae Daly (Bartonellaceae: Rhizobiales). Flea populations were maintained by membrane feeding on pathogen-free bloodmeals for up to 13 d post infection. Individual fleas and pools of flea feces were tested for the presence of Bartonella DNA using molecular methods (quantitative and conventional polymerase chain reaction [PCR]). The threshold number of Bartonellae required in the infectious bloodmeal for fleas to be detected as positive was 10⁶ colony-forming units per milliliter (CFU/ml). Individual fleas were capable of harboring infections for at least 13 d post infection and continuously excreted Bartonella DNA in their feces over the same period. This experiment demonstrated that X. cheopis are capable of acquiring and excreting B. elizabethae over several days.These results will guide future work to model and understand the role of X. cheopis in the natural transmission cycle of rodent-borne Bartonella species. Future experiments using this artificial feeding approach will be useful for examining the horizontal transmission of B. elizabethae or other rodent-associated Bartonella species to naïve hosts and for determining the viability of excreted bacteria.