Store-operated Orai1 channels mediate many critical cellular functions in animal cells ranging from gene expression to exocytosis. Activation of Orai1 channels by store depletion is driven by direct interactions between the Orai1 subunits with the endoplasmic reticulum Ca2+ sensor, STIM1, but how STIM1 binding transduces into opening of the Orai1 channel pore remains unclear. At the structural level, each Orai1 channel consists of six subunits with the transmembrane helices (TMs 1-4) of each subunit arranged in three concentric rings around a central aqueous pore formed by TM1. A prevailing model of channel gating postulates that STIM1 binding to the peripherally located Orai1 C-terminus is followed by a second, weaker STIM1 binding step to the centrally located N-terminus to drive pore opening. However, recent reports have cast doubt on the existence of a N-terminal STIM1 binding site, raising questions about how the gating signal is transmitted to the central pore. Here, we investigated the role of the transmembrane domains in this process and identified sixteen mutations in the non-pore lining TMs 2-4 that activate Orai1 channels in the absence of STIM1. Cysteine accessibility analysis and MD simulations indicate that the most robust of these activating mutations, H134S, induces rotation of the pore helix to move the side-chains of the gate formed by F99 aside, analogous to gating by STIM1. Further, atomic packing and mutational analysis revealed critical contact sites between TM1 and the TM2/3 helices, whose disruption by mutagenesis abrogated channel activation by STIM1 binding. We conclude that specific interactions between the transmembrane domains of Orai1 are critical for relaying the STIM1 activating signal from the C-terminus to the pore.