Orientia tsutsugamushi, the causative agent of scrub typhus, is an obligate intracellular parasite. Previously we have shown that it persistently infect the human endothelial cell line ECV304. In this study, we have investigated the mechanism of in vitro persistent infection, which could be maintained for over a year without addition of normal cells. The persistently infected cultures exhibited cyclic changes in the host cell number, which resulted in a net increase of cell number. Floating cells detached from the culture plate bottom were filled with rickettsial cells and lost normal morphology. Only part of attached EVC304 cells was infected with O. tsutsugamushi. Some of ECV304 cells that still attached to the bottom were free of rickettsial cells. Rickettsial cells in the floating cells could not effectively infect new EVC304 cells while those in the attached cell could infect new cells with high efficiency. Host cells that allowed vigorous rickettsial multiplication and resultantly lost viability, and low of infectivity of the bacteria in the dead cells might have allowed in vitro persistent infection of O. tsutsugamushi.