Conotoxins are marine peptide toxins from marine cone snails. The α-conotoxin RegIIA can selectively act on human (h) α3β4 nicotinic acetylcholine receptor (nAChR), and is an important lead for drug development. The high-resolution cryo-electron microscopy structure of the α3β4 nAChR demonstrates several carbohydrates are located near the orthosteric binding sites, which may affect α-conotoxin binding. Oligosaccharide chains can modify the physical and chemical properties of proteins by changing the conformation, hydrophobicity, quality and size of the protein. The purpose of this study is to explore the effect of oligosaccharide chains on the binding modes and activities of RegIIA and its derivatives at hα3β4 nAChRs. Through computational simulations, we designed and synthesized RegIIA mutants at position 14 to explore the importance of residue H14 to the activity of the peptide. Molecular dynamics simulations suggest that the oligosaccharide chains affect the binding of RegIIA at the hα3β4 nAChR through direct interactions with H14 and by affecting the C-loop conformation of the binding sites. Electrophysiology studies on H14 analogues suggest that in addition to forming direct interactions with the carbohydrates, the residue might play an important role in maintaining the conformation of the peptide. Overall, this study further clarifies the structure–activity relationship of α-conotoxin RegIIA at the hα3β4 nAChR and, also provides important experimental and theoretical basis for the development of new peptide drugs.