The structural mechanisms of α7 nicotinic receptor allosteric modulation and activation were investigated using high-resolution structures of α7-modulator complexes. These structures reveal partially overlapping binding sites but varying conformational states. Structural and computational analyses suggest that differences in modulator activity arise from the stable rotation of a channel gating residue out of the pore. Time-resolved cryo-EM reveals asymmetric state transitions in this homomeric channel, and allosteric agonists trigger a unique gating cycle. The study defines mechanisms of α7 allosteric modulation and activation, with implications across the pentameric receptor superfamily. The α7 nicotinic receptor is a pentameric ligand-gated ion channel involved in cholinergic signaling. Its unique physiological characteristics and implications in neurological disorders make it a promising therapeutic target. Positive allosteric modulators (PAMs) enhance neurotransmitter activation without activating the receptor, and their potentiation mechanisms remain unclear. The study presents high-resolution structures of α7-modulator complexes, revealing binding sites and conformational states. Structural analysis highlights three α7 residues (N213, M253, and A275) that likely contribute to modulator activity. Mutations in these residues affect modulator activity, indicating their importance in regulating PAM function. The study also shows that type I and type II PAMs stabilize distinct pore conformations, with type II PAMs extending the lifetime of the activated state by slowing desensitization. The results suggest that PAMs enhance α7 activity by stabilizing the L9' rotation out of the pore. Allosteric agonists (ago-PAMs) trigger a unique gating cycle, distinguishing them from traditional agonists. The study highlights the importance of L9' rotation in channel activation and the therapeutic potential of PAMs. The findings provide insights into the structural basis of modulator activity and the mechanisms of allosteric modulation in pentameric ion channels.The structural mechanisms of α7 nicotinic receptor allosteric modulation and activation were investigated using high-resolution structures of α7-modulator complexes. These structures reveal partially overlapping binding sites but varying conformational states. Structural and computational analyses suggest that differences in modulator activity arise from the stable rotation of a channel gating residue out of the pore. Time-resolved cryo-EM reveals asymmetric state transitions in this homomeric channel, and allosteric agonists trigger a unique gating cycle. The study defines mechanisms of α7 allosteric modulation and activation, with implications across the pentameric receptor superfamily. The α7 nicotinic receptor is a pentameric ligand-gated ion channel involved in cholinergic signaling. Its unique physiological characteristics and implications in neurological disorders make it a promising therapeutic target. Positive allosteric modulators (PAMs) enhance neurotransmitter activation without activating the receptor, and their potentiation mechanisms remain unclear. The study presents high-resolution structures of α7-modulator complexes, revealing binding sites and conformational states. Structural analysis highlights three α7 residues (N213, M253, and A275) that likely contribute to modulator activity. Mutations in these residues affect modulator activity, indicating their importance in regulating PAM function. The study also shows that type I and type II PAMs stabilize distinct pore conformations, with type II PAMs extending the lifetime of the activated state by slowing desensitization. The results suggest that PAMs enhance α7 activity by stabilizing the L9' rotation out of the pore. Allosteric agonists (ago-PAMs) trigger a unique gating cycle, distinguishing them from traditional agonists. The study highlights the importance of L9' rotation in channel activation and the therapeutic potential of PAMs. The findings provide insights into the structural basis of modulator activity and the mechanisms of allosteric modulation in pentameric ion channels.