Store-operated Ca2+entry (SOCE) is a universal Ca2+influx pathway that is important for the function of many cell types. SOCE is controlled by the interaction of the ER Ca2+sensor STIM1 with the plasma membrane Ca2+channel Orai1. S417 is located in the third coiled-coil (CC3) domain of the C-terminus of STIM1. We found that single-point mutation of this residue (S417G) abolished STIM1 C-terminus interactions with Orai1. Mutation of S417 also abolished CAD-Orai1 binding and Orai1 channel activation, eliminated STIM1 puncta formation, and co-localization with Orai1 and SOCE. 2-APB was found to restore the binding of the STIM1 C-terminus mutant (S417G) to Orai1 and dose-dependently activate Orai1 channel. Both CBD and NBD of Orai1 are required for 2-APB–induced coupling between the Orai1 and STIM1 C-terminus mutant (S417G) and CRAC channel activation. We also demonstrated that 2-APB led to delayed activation of Orai1-K85E channel, although Orai1-K85E obviously impairs 2-APB–induced STIM1 C-terminus mutant (S417G)–Orai1 coupling. Our results suggest S417 in the CC3 domain of STIM1 is essential for STIM1–Orai1 binding and CRAC channel activation.