Abstract: To explore the interaction between nicotine (NIC) of different configurations and biological proteins in human bodies, a variety of spectroscopic techniques were used to study the interaction mechanism and binding mode between the two isomers (S-type and R-type) of NIC and gastrin I Human (GIh) and the effect of NIC on GIh structure. The results showed that: 1) There were weaker bindings between the two isomers of NIC and GIh, in which hydrophobic interaction was the dominated one. The binding strength between S-NIC and GIh was slightly stronger than R-NIC. 2) Both isomers of NIC were sensitive to temperature. Raising temperature within a certain extent was beneficial to the binding between NIC and GIh. 3) The fluorescence quenching mechanisms of S- and R-NIC for GIh were the same, both were static quenching modes. The microenvironment around amino acid residues was changed to a certain extent when the two nicotine configurations acted on GIH. Based on circular dichroism(CD) results, the configuration inversion of NIC occurred while binding to GIh, which indicated that the binding between NIC and GIh might be induced by the inversion of NIC configuration.