Bartonella spp. are most often transmitted by arthropod vectors or animal bites and scratches. However, the vector species involved in the transmission of human bartonellosis remain poorly understood. This study investigated the presence of Bartonella in Triatoma rubrofasciata from Guangxi and Hainan provinces in China, evaluating its potential as a vector. Bartonella was identified in T. rubrofasciata samples through PCR amplification and sequencing of the ITS, gltA, and rpoB genes. The survival duration of Bartonella in triatomines, along with the potential for transovarial transmission was examined. Transmission experiments were conducted to determine whether T. rubrofasciata could transmit Bartonella to mice. Additionally, Bartonella spp. were also compared across rats, ticks, and cat fleas collected from the same regions. Results: Six Bartonella species were identified in T. rubrofasciata, including B. rochalimae, B. elizabethae, B. tribocorum, B. queenslandensis, B. silvatica, and B. coopersplainsensis. And the first three species are zoonotic. B. rochalimae and B. elizabethae were able to persist in T. rubrofasciata for at least eight weeks, although transovarial transmission of them was not observed. In comparison to rats, ticks, and cat fleas, T. rubrofasciata exhibited a higher diversity of Bartonella species. Laboratory experiments confirmed that B. elizabethae can infect mice through T. rubrofasciata bites or intraperitoneal injection of T. rubrofasciata feces. This study supports the hypothesis that T. rubrofasciata may serve as a vector for bartonellosis. These results broaden the current understanding of Bartonella transmission dynamics and highlight the potential role of triatomines in the spread of this disease.