Introduction: The aim of this study was to determine the effect of the continuous wave of condensation technique (CWCT) and the thermoplastic gutta-percha injection (TGI) technique on the messenger RNA (mRNA) expressions of heat shock proteins (HSPs) and mineralized tissue–associated proteins of the immortalized mouse cementoblasts (OCCM.30). Methods: Crowns of human premolar teeth with single and straight canals were removed. The root canals were prepared up to the ProTaper Next X5 file (Dentsply Maillefer, Ballaigues, Switzerland) in combination with 2 mL 2.5% sodium hypochlorite solution. Roots (12 ± 2 mm height) were sterilized (121°C for 20 minutes) and placed vertically to the cell culture dishes using a tissue culture insert by opening holes according to the root diameter after the removal of 1 mm from the apex for appropriate adaptation to the petri dish surfaces. Six groups were created: control 1 (without teeth), control 2 (with teeth), AH Plus (Dentsply DeTrey, Konstanz, Germany), single-cone obturation (SC), CWCT, and thermoplastic gutta-percha injection technique (TGI). The viability of the OCCM.30 cells was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell viability experiments at 24 and 96 hours. The mRNA expression of HSP27, HSP70, and HSP90 and mineralized tissue markers (bone sialoprotein, osteocalcin, runt-related transcription factor 2, type I collagen, and alkaline phosphatase) was evaluated by real-time polymerase chain reaction. Results: Reduced OCCM.30 cell viability was observed in all groups except the control groups. When the SC technique and CWCT and TGI groups were compared, it was observed that heat had a significant negative effect on cell viability (P <.05). A reduction in the mRNA expressions of HSP27, HSP70, and HSP90 was recognized in all test groups when compared with the control 1 group (P <.01). When the warm gutta-percha techniques were compared with the SC technique, a decrease in mRNA expression of HSP27 and HSP90 was noted (P <.01). The HSP70 transcript was similar in the CWCT group and the SC group. Higher HSP70 mRNA expression was observed in the TGI group compared with the SC group. In all groups except the control 1 group, bone sialoprotein, osteocalcin, runt-related transcription factor 2, type I collagen, and alkaline phosphatase mineralized tissue markers were affected, but this negative effect was higher in the heat-treated groups (P <.05). Conclusions: Within the limitations of this study, it was concluded that warm gutta-percha techniques reduced the mRNA expressions of the genes for HSPs and mineralized tissue–associated proteins of cementoblasts. Further animal studies are needed to clarify the effect of heat on the behavior of cementoblasts histologically in short- and long-term periods.