Tentonin 3 (TTN3/TMEM150C) is a mechanosensitive ion channel that responds to mechanical stimuli and exhibits distinct slow inactivation kinetics. It plays critical roles in mechanotransduction processes such as proprioception, blood pressure regulation, and insulin secretion. Structurally, TTN3 forms a tetramer with a predicted rectangular shape and a central pore. The conotoxin ρ-TIA and its synthetic analog, noxious mechanosensation blocker 1 (NMB-1), were initially developed to inhibit slowly adapting mechanically activated (MA) currents in dorsal root ganglion (DRG) neurons. Since TTN3 underlies these slowly adapting MA currents in DRG neurons, both NMB-1 and ρ-TIA were hypothesized to inhibit TTN3 activity. Indeed, NMB-1 strongly inhibited TTN3, whereas ρ-TIA had only a weak effect, and neither compound affected Piezo1. Alanine-scanning mutagenesis coupled with electrophysiological assays revealed that positively charged residues in NMB-1 are essential for its inhibitory function. Additionally, a glutamate residue (Glu126) near the TTN3 pore entrance was identified as critical for NMB-1’s inhibitory action, suggesting a key electrostatic interaction. Molecular dynamics simulations further support this electrostatic interaction between the peptide ligand and the channel protein. These findings offer mechanistic insight into the selective inhibition of TTN3 by NMB-1 and provide a foundation for developing therapeutic agents targeting TTN3-related channelopathies.