Tentonin 3/TMEM150C (TTN3) is a mechanically activated (MA) channel that opens rapidly in response to mechanical stimuli and exhibits slow inactivation. Although many MA channels are known to be modulated by forces arising from membrane lipids and cytoskeletal tethering proteins, the mechanisms governing TTN3’s mechanosensitivity remain poorly understood. Here, we report that the pharmacological disruption of the F-actin cytoskeleton or downregulation of focal adhesion genes significantly reduced TTN3 MA currents. Strengthening the F-actin cytoskeleton rescues robust TTN3 MA currents in Piezo1-deficient cells. Furthermore, we identify stomatin-like protein 3 (StomL3) as a key modulator of TTN3, which enhances membrane stiffness through cholesterol recruitment and physically associates with the serine-rich domain of the C-terminus in TTN3 to boost its mechanosensitivity. Our findings highlight that TTN3 mechanosensitivity highly depends on both cytoskeletal integrity and StomL3-dependent cell stiffness.