Stromal interaction molecule 1 (STIM1) stimulates calcium ion (Ca(2+)) entry through plasma membrane Orai1 channels in response to decreased Ca(2+) concentrations in the endoplasmic reticulum lumen. We identified an acidic motif within the STIM1 coiled-coil region that keeps its Ca(2+) activation domain [Ca(2+) release-activated Ca(2+) (CRAC) activation domain/STIM1-Orai activating region (CAD/SOAR)]-a cytoplasmic region required for its activation of Orai1-inactive. The sequence of the STIM1 acidic motif shows substantial similarity to that of the carboxyl-terminal coiled-coil segment of Orai1, which is the postulated site of interaction with STIM1. Mutations within this acidic region rendered STIM1 constitutively active, whereas mutations within a short basic segment of CAD/SOAR prevented Orai1 activation. We propose that the CAD/SOAR domain is released from an intramolecular clamp during STIM1 activation, allowing the basic segment to activate Orai1 channels. This evolutionarily conserved mechanism of STIM1 activation resembles the regulation of protein kinases by intramolecular silencing through pseudosubstrate binding.