The deformation to the brain that occurs during traumatic brain injury (TBI) results in a complex strain distribution throughout the brain tissue. Recently, many in vitro models of neuronal injury have been developed to simplify the mechanics which occur during TBI. We hypothesized that the type of mechanical insult imparted onto neurons would significantly alter both the mechanism and severity of the neuronal response to injury. In this study, primary cortical neurons were cultured on an elastic substrate and subjected to graded levels (0%, 10%, 30%, 50%) of either uniaxial (cells stretched in one direction only) or biaxial (cells simultaneously stretched in two directions) stretch. We found that neurons stretched in either injury paradigm exhibited immediate increases in intracellular free calcium ([Ca²⁺]_i), but the magnitude of the ([Ca²⁺]_i) rise was nearly an order of magnitude higher in biaxially stretched neurons compared to uniaxially stretched neurons. Moreover, while the ([Ca²⁺]_i) transient after uniaxial stretch was blocked with specific channel antagonists (APV, CNQX, nimodipine, TTX), a substantial ([Ca²⁺]_i) transient persisted in biaxially stretched neurons. We theorized that the additional calcium influx after biaxial stretch entered through nonspecific pores/tears formed in the membrane, since biaxially stretched neurons exhibited significant uptake of carboxyfluorescein, a molecule typically impermeant to cell membranes. Despite the large ([Ca²⁺]_i) transients, neither injury profile resulted in death within 24 h of injury. Interestingly, though, uniaxially stretched neurons exhibited enhanced [Ca+2]i influx following NMDA stimulation 24 h after trauma, compared to both control and biaxially stretched neurons. These data point out that the type of mechanical insult will influence the acute mechanisms of injury in vitro, can cause differences in the response to potential secondary excitotoxic injury mechanisms, and emphasizes the need to further study how these mechanical conditions can separately affect cell fate following mechanical injury.