STIM1 has been identified as a new warm sensor, but the exact molecular mechanism remains unclear. In this study, a variety of mutants of STIM1, Orai1 and Orai3 were generated. The single–cell calcium imaging and confocal analysis were used to evaluate the thermal sensitivity of the resulting STIM mutants and the interaction between STIM1 and Orai mutants in response to temperature. Our results suggested that the CC1–SOAR of STIM1 was a direct activation domain of temperature, leading to subsequent STIM1 activation, and the transmembrane (TM) region and K domain but not EF–SAM were needed for this process. Furthermore, both the TM and SOAR domains exhibited similarities and differences between STIM1–mediated thermal sensation and store–operated calcium entry (SOCE), and the key sites of Orai1 showed similar roles in these two responses. Additionally, the TM23 (comprising TM2, loop2, and TM3) region of Orai1 was identified as the key domain determining the STIM1/Orai1 thermal response pattern, while the temperature reactive mode of STIM1/Orai3 seemed to result from a combined effect of Orai3. These findings provide important support for the specific molecular mechanism of STIM1–induced thermal response, as well as the interaction mechanism of STIM1 with Orai1 and Orai3 after being activated by temperature.