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MicroRNAs (miRNAs) are important regulators of embryonic stem cell (ESC) biology, and their study has identified key regulatory mechanisms. To find novel pathways regulated by miRNAs in ESCs, we undertook a bioinformatics analysis of gene pathways differently expressed in the absence of miRNAs due to the deletion of <i>Dicer</i>, which encodes an RNase that is essential for the synthesis of miRNAs. One pathway that stood out was Ca²⁺ signaling. Interestingly, we found that <i>Dicer</i>^−/− ESCs had no difference in basal cytoplasmic Ca²⁺ levels but were hyperresponsive when Ca²⁺ import into the endoplasmic reticulum (ER) was blocked by thapsigargin. Remarkably, the increased Ca²⁺ response to thapsigargin in ESCs resulted in almost no increase in apoptosis and no differences in stress response pathways, despite the importance of miRNAs in the stress response of other cell types. The increased Ca²⁺ response in <i>Dicer⁻</i>^/<i>−</i> ESCs was also observed during purinergic receptor activation, demonstrating a physiological role for the miRNA regulation of Ca²⁺ signaling pathways. In examining the mechanism of increased Ca²⁺ responsiveness to thapsigargin, neither store-operated Ca²⁺ entry nor Ca²⁺ clearance mechanisms from the cytoplasm appeared to be involved. Rather, it appeared to involve an increase in the expression of one isoform of the IP₃ receptors (<i>Itpr2</i>). miRNA regulation of <i>Itpr2</i> expression primarily appeared to be indirect, with transcriptional regulation playing a major role. Therefore, the miRNA regulation of <i>Itpr2</i> expression offers a unique mechanism to regulate Ca²⁺ signaling pathways in the physiology of pluripotent stem cells.