OmIA, isolated from Conus omaria venom, is a potent antagonist at α7 nAChRs. We determined the co-crystal structure of OmIA with Lymnae stagnalis acetylcholine binding protein (Ls-AChBP). His5, Val10 and Asn11 responsible for the high potency of OmIA at α7 nAChRs. Remarkably, despite a competitive binding mode observed in the co-crystal structure, at α7 nAChRs and α3b4 nAChRs, OmIA and its analogues displayed insurmountable antagonism. At α7 nAChRs in the presence of type II positive allosteric modulators (PAMs), OmIA analogues with long side chain residues at position 10 (OmIA [V10Q] and OmIA [V10L]) appear as partial insurmountable antagonists. A "two-state, two-step" model was proposed to explain for this phenomenon. OmIA and other analogues were suggested to form a stable complex with the receptors that is slowly reversible to be occupied and stimulated by the agonist, while OmIA [V10Q] and OmIA [V10L] co-exist in a fast reversible/surmountable as well as a tight binding/insurmountable state. At PNU120596-α7 nAChRs, OmIA and analogues also display biphasic-inhibition behaviour with preference for low-affinity binding site at short preincubation time and high-affinity binding site at prolonged preincubation time. The complex pharmacological profile of OmIA at α7 nAChRs presented in this work emphasize α-conotoxins as a valuable tool to probe the pharmacological properties of nAChRs and may guide the development novel α7 modulators.