Diffuse axonal injury (DAI), a major component of traumatic brain injury, is associated with rapid deformation of brain tissue resulting in the stretching of neuronal axons. Focal axonal beading, which is the morphological hallmark of DAI pathology, leads to the disconnection of neurons from tissues and results in cell death. Our goal is to achieve a better understanding of neuronal tolerance and help predict the pathogenesis of DAI from mechanical loading to the head. In the present study, we developed an experimental model that subjected cultured neurons to uniaxial stretch by controlling the direction of axonal elongation with a microfluidic culture technique and examined the effect of strains along the axon on cell damage. Neurites from PC 12 cells that differentiate into neurons with structurally axon-like cylindrical protrusions by nerve growth factor were extended at 0°, 45°, and 90° relative to the tensile direction by using a fabricated polydimethylsiloxane (PDMS) piece with microgrooves in combination with a PDMS substrate. The morphology of the same neurites was observed before and after stretching with a strain of 0.22 at a strain rate of 27 s^-1. As a result, swellings along neurites oriented at 0° increased immediately following stretching and were sustained for 24 h. In contrast, swellings along neurites oriented at 45° and 90° transiently increased within 1 h following stretching. Although more ruptures were observed in neurites oriented at 0° and 90° than in those oriented at 45°, the number of neurites and cells did not differ among orientation conditions. These results suggest that the difference in strain along the axon induces axonal injuries differing in type and degree. They also suggest that the strain on the axon, rather than that on the cell-cultured substrate, is important for evaluating neuronal damage.