Stromal interaction molecule 1 (STIM1) stimulates calcium entry through plasma membrane Orai channel, which is critical for T cell activation and many cell processes. However, the molecular details of their interactions remain elusive. Here, we identified important regions in the N- and C-termini (NT and CT) of Orai1 and further analyzed the STIM1-Orai interactions using synthetic peptides derived from NT and CT of Orai1/Orai3 and purified fragments of STIM1. Our results provide the significant insight into CRAC channel gating by STIM1.