Resveratrol (RES) is a non-flavonoid polyphenols found in many plants, such as Vitaceae and Liliaceae. It is a natural active substance with variety biological and pharmacological functions and widely used in food and pharmaceutical field. Studies have shown that polyphenols had an interaction with digestive enzymes (such as pepsin, trypsin, etc.) in the process of digestion and absorption of organisms, resulting in changes in the biological activity of polyphenols and digestive enzymes and affecting the digestion and absorption of polyphenols and other nutrients. However, the mechanism of interaction between RES and pepsin (PEP) has rarely been reported. The attempt of this paper was to investigate the binding characteristics between RES and PEP at different temperatures by fluorescence spectroscopy, UV-Vis absorption spectroscopy, infrared spectroscopy (FT-IR) and molecular modeling technique. The experimental results provided important information for elucidating the action mechanism of RES and PEP. Fluorescence data revealed that the fluorescence intensity of PEP decreased regularly with the increase of RES concentration, indicating that RES had a fluorescence quenching effect on PEP. After RES was added, the UV-vis spectra of PEP changed significantly. The Kq value (the minimum quenching rate constant) at different temperatures were all much larger than 2.0×1010 L·mol-1·s-1(the maximum diffusion collision constant of the quenching agent on biological macromolecules). Moreover, Stern-Volmer quenching constant (KSV) gradually decreased with the increase in temperature. These results verified quenching mode between PEP and RES to be static. The value of the stoichiometric binding number approximately equals 1, suggesting that one molecule of RES combined with one molecule of PEP. The thermodynamic parameters indicated that RES could spontaneously bind with pepsin mainly through the hydrogen bonds and Van der Waals forces. Synchronous fluorescence and three-dimensional fluorescence results provided data concerning conformational and some micro-environmental changes of pepsin. According to the results from FTIR analyses of PEP, the content of β-sheet increased accompanying with significantly decrease of α-helix, and no obvious change of β-turn and random coil upon binding with RES. The presence of RES loosened the skeleton of pepsin. These secondary structure changes might lead to changes of the physiological function of pepsin, such as the enzyme activity. Finally, molecular docking further suggested that RES molecule binded within the active pocket of PEP mainly via the van der Waals forces and hydrogen bonds. There were the van der Waals forces between RES and residues Asp-32, Gly-34, Ser-35, Asn-37, Tyr-75, Gly-76, Thr-77, Ile-128, Ala-130 and Gly-217 of PEP, super conjugation between RES and residues Ile-128 and Asp-215 of PEP, and hydrogen bonds between RES and Ser-36, Asn-37, Ile-128 and Thr-218 of PEP. Various forces make RES and PEP form a more stable complex.