Ischemic brain injury represents a major cause of death and disability. Developing targeted therapies for neuroprotection requires increased understanding of the molecular mechanisms of neuronal injury following ischemia. Both disruption of Ca2+ homeostasis and pathological activation of proteases are believed to play causal roles in delayed neuronal death after ischemia-reperfusion. Presented here are data supporting a novel role for proteolysis of an intracellular Ca2+ release channel, inositol 1,4,5-trisphosphate receptor (InsP3R), in ischemic brain injury. We identified a unique calpain cleavage site in the type 1 InsP3R (InsP3R1) and utilized a recombinant truncated form of the channel (capn-InsP3R1) to investigate the functional consequences of InsP3R1 proteolysis. Using a combination of single-channel electrophysiology and single-cell Ca2+ imaging, we determined that capn-InsP3R1 has InsP3-independent gating and constitutive channel activity. This constitutive channel activity decreased Ca2+ content of intracellular stores in Neuro-2A cells by increasing endoplasmic reticulum (ER) Ca2+ leak. Additionally, capn-InsP3R1 compromised ER Ca2+ buffering capacity in primary cortical cultures, leading to decreased neuronal viability and enhanced sensitivity to excitotoxic injury. Using stereotaxic intracerebral injection of viral vectors, we transduced neurons in vivo with capn-InsP3R1 and observed spontaneous degeneration of subpopulations of neurons in the hippocampus. Together, these results reveal a previously unknown role of calpain-cleaved InsP3R1 in disruption of intracellular Ca2+ homeostasis and neuronal death. Importantly, we also provide evidence of calpain-mediated proteolysis of InsP3R1 in neurons in the cerebellum after ischemic brain injury. The findings presented here provide the first functional studies of calpain-cleaved InsP3R1, and advance our understanding of the pathological role of the cleaved channel in neurodegeneration. Inhibiting Ca2+ release through calpain-cleaved InsP3R1 may emerge as a novel therapeutic strategy for intervention in ischemic brain injury and other neurodegenerative diseases associated with disruption of Ca2+ homeostasis.