Background and purpose: Store-operated Ca2+ entry (SOCE) is important for the function of many cell types. It is controlled by the interaction between ER Ca2+ sensor STIM1 and the plasma membrane Ca2+ channel Orai1. CAD of STIM1 is required for SOCE. It contains two putative coiled-coil regions (CC2 and CC3). The role of CC3 remains to be elucidated. Experimental approach: various plasmids carrying different fluorescent protein genes were constructed for better understanding the influence of S417G mutation in CC3 on SOCE activation; Confocal imaging system, calcium imaging technique and FRET technique were employed to examine the actions of 2-APB on the interaction between STIM1 C terminus and Orai1. Key results: Single-point mutation of the residue (S417G) abolishes 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 were found to restore the binding of STIM1 C-terminus mutant(S417G) to Orai1 and dose-dependently activated Orai1 channel. Both CBD and NBD of Orai1 is required for 2-APB-induced coupling between Orai1 and STIM1 C terminus mutant(S417G) and CRAC channel activation. We also demonstrated 2-APB lead to delayed activation of Orai1-K85E channel, although Orai1-K85E obviously impair 2-APB-induced STIM1 C-terminus mutant(S417G)-Orai1 coupling. Conclusions and implications: Our data suggest that S417 in the CC3 domain of STIM1 is critical for STIM1-Orai1 binding and CRAC channel activation. We also proposed experimental models of combined STM1 or Orai1 mutants with 2-APB to better understand the activation mechanism of CRAC.