The interaction of the agonist JN403 with the human (h) α7 nicotinic acetylcholine receptor (AChR) was compared to that for the competitive antagonist methyllycaconitine (MLA). The receptor selectivity of JN403 was studied on the hα7, hα3β4, and hα4β2 AChRs. The results established that the cationic center and the hydrophobic group found in JN430 and MLA are important for the interaction with the AChRs. MLA preincubation inhibits JN403-induced Ca2+ influx in GH3-hα7 cells with a potency 160-fold higher than that when MLA is co-injected with JN403. The most probable explanation, based on our dynamics results, is that MLA (more specifically the 3-methyl-2,5-dioxopyrrole ring and the B-D rings) stabilizes the resting conformational state. The order of receptor specificity for JN403 is as follows: hα7 > hα3β4 (∼40-fold) > hα4β2 (∼500-fold). This specificity is based on a larger number of hydrogen bonds between the carbamate group (another pharmacophore) of JN403 and the hα7 sites, the electrostatic repulsion between the positively charged residues around the hα3β4 sites and the cationic center of JN403, fewer hydrogen bonds for the interaction of JN403 with the hα3β4 AChR, and an unfavorable van der Waals interaction between JN403 and the α4-β2 interface. The higher receptor specificity for JN403 could be important for the treatment of α7-related disorders, including dementias, pain-related ailments, depression, anxiety, and wound healing.