We previously reported that TRPV2 can be activated by mechanical stimulation which enhances axonal outgrowth in developing neurons (Shibasaki et al., J. Neurosci. 2010). However, the molecular mechanisms governing the contribution of TRPV2 activation to axonal outgrowth remain unclear. In this study, we examined this mechanism. Overexpression of TRPV2 enhanced axonal outgrowth in a mechanical stimulus-dependent manner. Accumulation of TRPV2 at the cell surface was 4-fold greater in the growth cone compared to the soma. In the growth cone, TRPV2 is not static, but dynamically accumulates (within ~100 msec) to the site of mechanical stimulation. The dynamic and acute clustering of TRPV2 can enhance very weak mechanical stimuli through the focal accumulation of TRPV2. Focal application of mechanical stimuli dramatically increased growth cone motility and caused actin reorganization through the activation of TRPV2. We also found that TRPV2 physically interacts with actin, and changes in the actin cytoskeleton are required for its activation. Here, we demonstrated for the first time that TRPV2 clustering is induced by mechanical stimulation generated by axonal outgrowth, and TRPV2 activation is triggered by actin rearrangements resulting from mechanical stimulation. Moreover, TRPV2 activation enhances growth cone motility and actin accumulation to promote axonal outgrowth.