Nicotinic acetylcholine receptors (nAChRs) are heteropentameric ligand-gated ion channels that mediate excitatory neurotransmission at the neuromuscular junction (NMJ) and other peripheral and central synapses. At the NMJ, acetylcholine receptors (AChRs) are constantly exposed to mechanical stress resulting from muscle contraction. It is therefore of interest to understand if their function is influenced by mechanical stimuli. In this study, patch-clamp recordings showed that AChR channel activity was enhanced upon membrane stretching in both cultured Xenopus muscle cells and C2C12 myotubes. To examine how this property is physiologically regulated, effects of membrane-intrinsic and membrane-extrinsic factors on AChRs expressed in HEK293T cells were studied. As in muscle cells, AChR single channel currents recorded under cell-attached configuration were significantly increased-without change in current amplitude-when negative pressure was applied through the patch pipette. GsMTx-4, a peptide toxin that blocks mechanically activated cation channels, inhibited this effect on AChRs. The mechanosensitivity decreased when cells were treated with MβCD, latrunculin A or cytochalasin D, but increased when exposed to lysophosphatidylcholine, indicating contributions from both membrane lipids and the cytoskeleton. Rapsyn, which binds to AChRs and mediates their cytoskeletal interaction in muscle, suppressed AChR mechanosensitivity when co-expressed in HEK293T cells, but this influence of rapsyn was impaired following the deletion of rapsyn's AChR-binding domain or upon cytoskeletal disruption by cytochalasin D. These results suggest a mechanism for regulating AChR's mechanosensitivity through its cytoskeletal linkage via rapsyn, which may serve to protect the receptors and sarcolemmal integrity under high mechanical stress encountered by the NMJ.