In cerebellar Purkinje cells (PCs), the metabotropic glutamate receptor type 1 (mGluR1) is crucial for cerebellar function. Synaptic activation of mGluR1 is followed by InsP3 receptor-dependent Ca2+ release from endoplasmic reticulum (ER) Ca2+ stores and a slow EPSC (sEPSC) mediated by the transient receptor potential channel TRPC3. The intracellular mechanism of TRPC3 activation is unknown as is how ER Ca2+ content is maintained through many cycles of synaptic activation. In non-excitable cells TRPC channels are known to interact with the stromal interaction molecules (STIM1 and STIM2), which are also critical for Ca2+ store replenishment. Here we tested the hypothesis that STIM proteins may be involved in TRPC3 activation in central neurons. Values are presented as means ± SEM, compared by Student’s t-test. Quantitative single cell RT-PCR analysis showed that expression of STIM1 in PCs is ten times greater than that of its homolog STIM2 (103±71 vs. 11±8 copies mRNA/cell of Stim1 and Stim2 from n=23 and 14 cells, respectively, p<0.01). Using whole-cell recordings and confocal Ca2+ imaging in acute cerebellar slices from newly generated PC-specific STIM1/2 double-knockout (STIM1/2pdko) mice (C57/BL6 background, anesthetization by CO2 inhalation, immediate decapitation), we found that mGluR1-mediated Ca2+ release and the TRPC3-sEPSC evoked by repetitive parallel fibre stimulation (5 x 10V, 10 μM CNQX) are largely abolished in the absence of STIM1 and STIM2 (15.1±3.3 vs 89.1±15.9 pA; ΔF/F =34.1±8.3 vs 162.6±18.0 %, n=68 vs 32 inputs, p<0.01, respectively). Comparison with mice deficient in STIM1 alone (STIM1pko mice) showed that of the two homologs STIM1 has the primary role for mGluR1-dependent signaling in PCs. Synaptic stimulation-evoked sEPSCs and Ca2+ signals had mean amplitudes of 5±3 pA and ΔF/F = 2±1 %, respectively (n=21 inputs). We tested the function of ER Ca2+ stores specifically in the absence of STIM1. In control mice, Ca2+ release signals evoked by local application of the group I mGluR-specific agonist DHPG were abolished when depleting Ca2+ stores by switching to Ca2+ free extracellular perfusion media and recovered within a few minutes after returning to Ca2+ containing media. In STIM1pko mice, DHPG applications completely failed to evoke Ca2+ transients before, during and after the perfusion with Ca2+ free extracellular media. Interestingly, DHPG applications evoked both Ca2+ signals and TRPC3-mediated inward currents in STIM1pko mice immediately (4s) following Ca2+ influx through voltage-gated Ca2+ channels evoked by a short (1s) depolarization but failed to be effective at later time points. These results demonstrate that STIM1 is a powerful regulator of the ER store Ca2+ content in mammalian neurons. Furthermore, they identify STIM1 as a messenger that couples mGluR1 and TRPC3 in cerebellar PCs through the regulation of intracellular Ca2+.