Objective To discover novel conopeptides which are the antagonists of neuronal nicotinic acetylcholine receptors (nAChRs) in order to contribute to the development of novel analgesic drugs and neuropharmacological probes.Methods Based on the conserved untranslated region and intron of A-superfamily conotoxins,a novel α-conotoxin Lt1.1 was cloned from Conus litteratus.The peptide-resin was synthesized using the solid-phased method and was cleaved.The resulting linear peptide was oxidized by air to give the product containing disulfide bridges.The folding product was finally purified by HPLC.The disulfide bond connectivity was determined using the two-step oxidative folding methods.The cRNA of rat nAChRs was expressed on the membrane of Xenopus oocyte.Membrane currents were recorded using the two electrode voltage-clamp technique.Results A novel α-conotoxin designated as Lt1.1(GCCSHPACNVNNPDIC-NH2) was cloned and its disulfide connectivity was C1-C3,C2-C4.Lt1.1 selectively inhibited the α3β2 and α3β4 nAChRs with an IC50 of 166.76 and 190.00 nmol/L,respectively.Conclusion Lt1.1 is a novel 4/7 α-conotoxin that selectively targets α3β2 and α3β4 nAChRs.