Severe hyperbilirubinemia leads to bilirubin encephalopathy in neonates, with irreversible neurological sequelae. We investigated the neuronal vulnerability to unconjugated bilirubin (UCB) toxicity. The calcium (Ca2+) homeostasis is crucial for neuron survival. Ca2+ release from endoplasmic reticulum (ER) during ER-stress can lead to apoptosis trough Caspase-12 activation. By live Ca2+ imaging we monitored Ca2+ signals in hippocampal neuroglia cells exposed to UCB doses, showing the ability of UCB to alter intracellular Ca2+ homeostasis. The contribution of intracellular Ca2+ stores and the activation of proteins involved in the apoptotic Ca2+ signaling were also assessed. Thapsigargin, specific inhibitor of Sarco/endoplasmic reticulum ATPase pumps, significantly reduced the duration of Ca2+ oscillation associated with UCB exposure indicating that UCB strongly interfered with the reticulum Ca2+ stores. Contrarily, in pure astrocyte cultures, spontaneous Ca2+ transient duration was not altered by UCB. The protein content of GRP78, AT6, CHOP, Calpain and Caspase-12 treated with UCB was twofold higher compared to controls. Ca2+-dependent Calpain and Caspase-12 induction by UCB were significantly reduced when cells were pretreated with the ER-stress inhibitor 4-PBA. We showed the direct interference of UCB with neuronal intracellular Ca2+ dynamics, suggesting ER Ca2+ stores as a primary target of UCB toxicity with the activation of the apoptotic ER-stress-dependent pathway.