ABSTRACTTheRhabdochlamydiaceaefamily is a recent addition to theChlamydiaephylum. Its members were discovered in cockroaches and woodlice, but recent metagenomics surveys demonstrated the widespread distribution of this family in the environment. It was, moreover, estimated to be the largest family of theChlamydiaephylum based on the diversity of its 16S rRNA encoding gene. Unlike mostChlamydia-like organisms, noRhabdochlamydiaceaemember could be cultivated in amoebae, and its host range remains unknown. We tested the permissivity of various mammalian and arthropod cell lines to determine the host range ofRhabdochlamydia porcellionis, the only cultured representative of this family. While growth could initially be obtained only in the Sf9 cell line, lowering the incubation temperature of the mammalian cells from 37°C to 28°C allowed the growth ofR. porcellionis. Furthermore, a 6-h exposure to 37°C was sufficient to irreversibly block the replication ofR. porcellionis, suggesting that this bacterium either lost or never acquired the ability to grow at 37°C. We next sought to determine if temperature would also affect the infectivity of elementary bodies. Although we could not purify enough bacteria to reach a conclusive result forR. porcellionis,our experiment showed that the elementary bodies ofChlamydia trachomatisandWaddlia chondrophilalose their infectivity faster at 37°C than at room temperature. Our results demonstrate that members of theChlamydiaephylum adapt to the temperature of their host organism and that this adaptation can in turn restrict their host range.IMPORTANCETheRhabdochlamydiaceaefamily is part of theChlamydiae, a phylum of bacteria that includes obligate intracellular bacteria sharing the same biphasic developmental cycle. This family has been shown to be highly prevalent in the environment, particularly in freshwater and soil, and despite being estimated to be the largest family in theChlamydiaephylum is only poorly studied. Members of theRhabdochlamydiaceaehave been detected in various arthropods like ticks, spiders, cockroaches, and woodlice, but the full host range of this family is currently unknown. In this study, we showed thatR. porcellionis, the only cultured representative of theRhabdochlamydiaceaefamily, cannot grow at 37°C and is quickly inactivated at this temperature. A similar temperature sensitivity was also observed for elementary bodies of chlamydial species adapted to mammals. Our work demonstrates that chlamydiae adapt to the temperature of their reservoir, making a jump between species with different body temperatures unlikely.