We have previously reported that transient receptor potential vanilloid 2 (TRPV2) can be activated by mechanical stimulation, which enhances axonal outgrowth in developing neurons; however, the molecular mechanisms that govern the contribution of TRPV2 activation to axonal outgrowth remain unclear. In the present study, we examined this mechanism by using PC12 cells as a neuronal model. 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 with the soma. In the growth cone, TRPV2 is not static, but dynamically accumulates (within ∼100 ms) to the site of mechanical stimulation. The dynamic and acute clustering of TRPV2 can enhance very weak mechanical stimuli via focal accumulation of TRPV2. Focal application of mechanical stimuli dramatically increased growth cone motility and caused actin reorganization via activation of TRPV2. We also found that TRPV2 physically interacts with actin and that changes in the actin cytoskeleton are required for its activation. Here, we demonstrated for the first time to our knowledge that TRPV2 clustering is induced by mechanical stimulation generated by axonal outgrowth and that TRPV2 activation is triggered by actin rearrangements that result from mechanical stimulation. Moreover, TRPV2 activation enhances growth cone motility and actin accumulation to promote axonal outgrowth. —Sugio, S., Nagasawa, M., Kojima, I., Ishizaki, Y., Shibasaki, K. Transient receptor potential vanilloid 2 activation by focal mechanical stimulation requires interaction with the actin cytoskeleton and enhances growth cone motility. FASEB J. 31, 1368–1381 (2017) www.fasebj.org