Thein vivotemperature can vary according to the host tissue and the response to infection.Streptococcus pneumoniaehas evolved mechanisms to survive these temperature differences, but neither the consequences of different temperatures for pneumococcal phenotype nor the genetic basis of thermal adaptation are known in detail. In our previous study [16], we found that CiaR, which is a part of two-component regulatory system CiaRH, as well as 17 genes known to be controlled by CiaRH, were identified to be differentially expressed with temperature. One of the CiaRH-regulated genes shown to be differentially regulated by temperature is for the high-temperature requirement protein (HtrA), coded by SPD_2068 (htrA). In this study, we hypothesized that the CiaRH system plays an important role in pneumococcal thermal adaptation through its control overhtrA. This hypothesis was evaluated by testing strains mutated or overexpressingciaRand/orhtrA, inin vitroandin vivoassays. The results showed that in the absence ofciaR, the growth, haemolytic activity, amount of capsule and biofilm formation were considerably diminished at 40 °C only, while the cell size and virulence were affected at both 34 and 40 °C. The overexpression ofhtrAin the ∆ciaRbackground reconstituted the growth at all temperatures, and the haemolytic activity, biofilm formation and virulence of ∆ciaRpartially at 40 °C. We also showed that overexpression ofhtrAin the wild-type promoted pneumococcal virulence at 40 °C, while the increase of capsule was observed at 34 °C, suggesting that the role ofhtrAchanges at different temperatures. Our data suggest that CiaR and HtrA play an important role in pneumococcal thermal adaptation.