BACKGROUND: Zein is commonly used to construct food flavonoid delivery systems. This study investigated the effect and mechanism of zein on the digestive stability of five citrus flavonoids, namely hesperetin (HET), hesperidin (HED), neohesperidin (NHD), naringenin (NEN), and naringin (NIN).RESULTS: Zein enhanced the digestive stability of the five citrus flavonoids, especially that of HET and NEN, during digestion in the stomach and small intestine. Fluorescence spectroscopy results suggested that citrus flavonoids spontaneously quenched the endogenous fluorescence of zein in static quenching mode. The binding of HET, HED and NHD to zein was driven respectively by electrostatic, hydrophobic and electrostatic interaction. However, Van der Waals' force and hydrogen (H)-bond interaction represented the primary driving force for binding NEN, and NIN to zein to form complexes. The binding of the five citrus flavonoids to zein also caused a diverse bathochromic shift in ultraviolet absorbance. Analysis using Fourier-transform infrared and Raman spectroscopy revealed that the binding behavior of the five citrus flavonoids had different effects on changes in the secondary structures, disulfide bonds, and tyrosine exposure of zein. The results were also partially verified by molecular dynamic simulation. CONCLUSIONS: Zein enhanced the digestive stability of the five citrus flavonoids via different binding interactions that was due to the difference in molecular structure of citrus flavonoids.