Bartonella henselae is a slow-growing, Gram-negative bacterium that causes cat scratch disease in humans. A transstadial transmission of the bacteria from larvae to nymphs of Rhipicephalus sanguineus sensu lato (s.l.) ticks, suspected to be a potential vector of the bacteria, has been previously demonstrated. The present study aims to investigate transovarial transmission of B. henselae from R. sanguineus s.l. adults to their instars. Adult ticks (25 males and 25 females) were fed through an artificial feeding system on B. henselae-infected goat blood for 14 days, and 300 larvae derived from the experimentally B. henselae-infected females were fed on noninfected goat blood for 7 days. Nested PCR and culture were used to detect and isolate B. henselae in ticks and blood samples. Bartonella henselae DNA was detected in midguts, salivary glands, and carcasses of the semi-engorged adults and pooled tick feces (during feeding and post-feeding periods). After the oviposition period, B. henselae DNA was detected in salivary glands of females (33.3%), but not in pooled eggs or larvae derived from the infected females. However, B. henselae DNA was detected by nested PCR from the blood sample during larval feeding, while no viable B. henselae was isolated by culture. According to our findings, following infected blood meal, B. henselae could remain in the tick midguts, move to other tissues including salivary glands, and then be shed through tick feces with limited persistency. The presence of bacterial DNA in the blood during larval feeding shows the possibility of transovarial transmission of B. henselae in R. sanguineus s.l. ticks.