Alterations in the expression, molecular composition, and localization of voltage-gated sodium channels play major roles in a broad range of neurological disorders. Recent evidence identifies sodium channel proteolysis as a key early event after ischemia and traumatic brain injury, further expanding the role of the sodium channel in neurological diseases. In this study, we investigate the protease responsible for proteolytic cleavage of voltage-gated sodium channels (NaChs). NaCh proteolysis occurs after protease activation in rat brain homogenates, pharmacological disruption of ionic homeostasis in cortical cultures, and mechanical injury using an in vitro model of traumatic brain injury. Proteolysis requires Ca²⁺ and calpain activation but is not influenced by caspase-3 or cathepsin inhibition. Proteolysis results in loss of the full-length α-subunits, and the creation of fragments comprising all domains of the channel that retain interaction even after proteolysis. Cell surface biotinylation after mechanical injury indicates that proteolyzed NaChs remain in the membrane before noticeable evidence of neuronal death, providing a mechanism for altered action potential initiation, propagation, and downstream signaling events after Ca²⁺ elevation.