Since traumatic axonal injury (TAI) is implicated as a prominent pathology of concussion, we examined potential sex differences in axon structure and responses to TAI. Rat and human neurons were used to develop micropatterned axon tracts in vitro that were genetically either male or female. Ultrastructural analysis revealed for the first time that female axons were consistently smaller with fewer microtubules than male axons. Computational modeling of TAI showed that these structural differences place microtubules in female axons at greater risk of failure during trauma under the same applied loads than in male axons. Likewise, in an in vitro model of TAI, dynamic stretch-injury to axon tracts induced greater pathophysiology of female axons than male axons, including more extensive undulation formations resulting from mechanical breaking of microtubules, and greater calcium influx shortly after the same level of injury. At 24 h post-injury, female axons exhibited significantly more swellings and greater loss of calcium signaling function than male axons. Accordingly, sexual dimorphism of axon structure in the brain may also contribute to more extensive axonal pathology in females compared to males exposed to the same mechanical injury.