Purpose <p>Tunneling nanotubes (TNTs) are membrane-based intercellular structures that support long-range communication between spatially separated cells. Although TNT-associated electrical responses have been observed under mechanical stimulation, whether these responses are linked to changes in cellular mechanical behavior remains unclear.</p> Methods <p>Mechanical stimulation was applied to one cell within TNT-connected C2C12 pairs. Traction force and cell spreading area in the connected cell were evaluated under different pharmacological conditions to investigate the involvement of calcium signaling, calcium channels, gap junction communication, and cellular contractility.</p> Results <p>Mechanical stimulation increased traction force and reduced cell spreading area in TNT-connected cells. These responses were suppressed by calcium depletion, inhibition of T-type calcium channels, or blockade of gap junction communication, but were not significantly affected by inhibition of L-type calcium channels.</p> Conclusions <p>These findings support a calcium-dependent association between TNT-mediated intercellular signaling and biomechanical regulation in connected C2C12 cells.</p>