The commonly used general anesthetic isoflurane induces widespread neurodegeneration in the developing mammalian brain through poorly understood mechanisms. We have investigated whether excessive Ca2+ release from the endoplasmic reticulum via overactivation of inositol 1,4,5-trisphosphate receptors (InsP3Rs) is a contributing factor in such neurodegeneration in rodent primary cultured neurons and developing rat brain. We also investigated the correlation between isoflurane exposure and cognitive decline in rats at 1 month of age. Our results show that isoflurane increases cytosolic calcium in the primary cortical neurons through release from the endoplasmic reticulum and influx from the extracellular space. Pharmacological inhibition of InsP3R activity and knockdown of its expression nearly abolishes the isoflurane-mediated elevation of the cytosolic calcium concentration and cell death in rodent primary cortical and hippocampal neurons. Inhibition of InsP3R activity by its antagonist xestospongin C significantly inhibits neurodegeneration induced by isoflurane at clinically used concentration in the developing brain of postnatal day 7 rats. Moreover, our results show that isoflurane activates beta-site amyloid beta precursor protein-cleaving enzyme via activation of the InsP3R. We also noted that mice exposed to isoflurane during early postnatal development showed transient memory and learning impairments, which did not correlate well with the noted neuropathological defects. Taken together, our results suggest that Ca2+ dysregulation through overactivation of the InsP3R may be a contributing factor in the mechanism of isoflurane-induced neurodegeneration in rodent neuronal cell culture and during brain development.