Acute axonal shear and stretch in the brain induces an evolving form of axonopathy and is a major cause of ongoing motor, cognitive and emotional dysfunction. We have utilized an in vitro model of mild axon bundle stretch injury, in cultured primary cortical neurons, to determine potential early critical cellular alterations leading to secondary axonal degeneration. We determined that transient axonal stretch injury induced an initial acute increase in intracellular calcium, principally derived from intracellular stores, which was followed by a delayed increase in calcium over 48 h post-injury (PI). This progressive and persistent increase in intracellular calcium was also associated with increased frequency of spontaneous calcium fluxes as well as cytoskeletal abnormalities. Additionally, at 48 h post-injury, stretch-injured axon bundles demonstrated filopodia-like sprout formation that preceded secondary axotomy and degeneration. Pharmacological inhibition of the calcium-activated phosphatase, calcineurin, resulted in reduced secondary axotomy (p < 0.05) and increased filopodial sprout length. In summary, these results demonstrate that stretch injury of axons induced an initial substantial release of calcium from intracellular stores with elevated intracellular calcium persisting over 2 days. These long-lasting calcium alterations may provide new insight into the earliest neuronal abnormalities that follow traumatic brain injury as well as the key cellular changes that lead to the development of diffuse axonal injury and secondary degeneration.