Oxytetracycline (OTC) is a kind of widely used veterinary drugs. The residue of OTC in the environment (e.g., animal food, soils, surface, and groundwater) is potentially harmful. In this article, the binding mode of OTC with trypsin was investigated using spectroscopic and molecular docking methods. OTC can interact with trypsin with one binding site to form OTC-trypsin complex, resulting in inhibition of trypsin activity and change of the secondary structure and the microenvironment of the tryptophan residues of trypsin. After elimination of the inner filter effect, the association constant, K, was calculated to be K-290K = 1.36 x 10(5) L mol(-1) K-298K = 7.30 x 10(4) L mol(-1) and K-307K = 3.58 x 10(4) L mol(-1) at three different temperatures. The calculated thermodynamic parameters (negative values of Delta H-O and Delta S-O) indicated that van der Waals interactions and hydrogen bonds play a major role during the interaction. The molecular docking study revealed that OTC bound into the S I binding pocket, which illustrates that the trypsin activity was competitively inhibited by OTC, in accordance with the conclusion of the trypsin activity experiment. This work establishes a new strategy to probe the toxicity of OTC and contributes to clarify its molecular mechanism of toxicity in vivo. The combination of spectroscopic and molecular docking methods in this work can be applied to investigate the potential enzyme toxicity of other small organic pollutants and drugs.