The endoplasmic reticulum (ER) Ca(2+) sensor stromal interaction molecule 1 (STIM1) binds and activates plasma membrane localized Orai1 channels, providing a Ca²⁺ influx pathway that is essential for proper immune cell function (store operated calcium entry, SOCE). Careful titration of the protein concentration of STIM1 and Orai1 thus determines the amount of Ca²⁺ influx and its subsequent downstream effects. Because changes in the degree and quality of glycosylation can also affect immune cell development and function, we investigated the impact of altered glycosylation on SOCE. Indeed, inhibition of oligosaccharyltransferases increased SOCE in Jurkat T cells. To delineate the contribution of Orai1 and STIM1, we mutated their potential N-glycosylation sites. Conventional replacement of the consensus asparagines by glutamines in STIM1 (N/Q) led to a reduction in Orai1 mediated currents. Other amino acid substitutions at the same positions led to variable degrees of current modification with one mutation leading to significantly increased current sizes. Interestingly, this mutation correlated with a change in Orai1 protein concentration and led to a change in the STIM1:Orai1 stoichiometry towards an optimized ratio for Orai1 activation. Noise analyses of Orai1 mediated currents revealed an increase in the number of active channels, with little change in open probability or single channel conductance. The phenotype of the mutant could only be partially mimicked by alteration of wildtype protein ratios between Orai1 and STIM1, pointing to an additional influence of the mutation on the EF-SAM domain stability or function. Our current data suggests that our gain-of-function STIM1 mutant may overcome the negative cooperativity which limits interaction of wildtype STIM1 with Orai1.