Bartonella are emerging and re-emerging pathogens affecting humans and a wide variety of animals including rodents. Horizontal transmission of Bartonella species by different hematophagous vectors is well acknowledged but vertical transmission (from mother to offspring) is questionable and was never explored in fleas. The aim of this study was to investigate whether the rodent flea, Xenopsylla ramesis, can acquire native Bartonella from wild rodents and transmit it transovarially. For this aim, Bartonella-free laboratory-reared X. ramesis fleas were placed on six naturally Bartonella-infected rodents and six species-matched Bartonella-negative rodents (three Meriones crassus jirds, two Gerbillus nanus gerbils and one Gerbillus dasyurus gerbil) for 7 days, 12–14 h per day. The fleas that were placed on the Bartonella-positive rodents acquired four different Bartonella genotypes. Eggs and larvae laid and developed, respectively, by fleas from both rodent groups were collected daily for 7 days and molecularly screened for Bartonella. All eggs and larvae from both groups were found to be negative for Bartonella DNA. Interestingly, two of five gut voids regurgitated by Bartonella-positive fleas contained Bartonella DNA. The naturally infected rodents remained persistently infected with Bartonella for at least 89 days suggesting their capability to serve as competent reservoirs for Bartonella species. The findings in this study indicate that X. ramesis fleas can acquire several Bartonella strains from wild rodents but cannot transmit Bartonella transovarially.