The aim of this study was to investigate the inhibitory mechanism of oenothein B (OeB), a unique oligomer ellagitannin with a rigid structure, on porcine trypsin using fluorescence spectroscopy, isothermal titration calorimetry (ITC), circular dichroism (CD) and molecular docking. Trypsin activity was strongly inhibited by OeB in a competitive way. Fluorescence quenching of trypsin by OeB was a static quenching. The CD spectra showed that binding of OeB to trypsin altered trypsin's conformation. The ITC and docking studies revealed that the inhibitory mechanism of OeB occurred via binding to the interior hydrophobic groups of trypsin and the formation of hydrogen bonds with trypsin through binding to the amino acid residues Asn97, His573, Ser195 and Gln192. This study provides a theoretical and computational basis for the precise control of trypsin in food industry. Based on the results, OeB may be used in food technology research as novel bioactive trypsin inhibitor.