2-Aminoethyldiphenyl borate (2-APB) is the most commonly used pharmacological agent in the study of calcium release-activated channels (CRACs); however, its inhibitory mechanism to CRACs remains unclear. To address this issue, we systematically employed confocal imaging, dual-wavelength excitation photometry and FRET to examine the effects of 2-APB on the dynamic activities and function of STIM1 and Orai1, two key components of CRACs. Imaging results support that there are two signaling pathways (Orai1-independent and Orai1-dependent) for the formation of STIM1 puncta. 2-APB could dose dependently block Orai1-independent but not Orai1-dependent STIM1 puncta formation, despite its obvious inhibition effect on storeoperated Ca2+ entry (SOCE). In addition, we found that although 2-APB could not visibly alter near plasma membrane CAD-eYFP localization, it could completely block CAD-YFP-induced constitutive Ca2+ entry and promote the interaction between Orai1 and CAD by FRET measurements. Therefore, we proposed that inhibitory action of 2-APB on SOCE might attribute to its direct inhibitory effects on Orai1 channel itself, but not the interference on puncta formation between STIM1 and Orai1.