Being the first-degree toxic pollutants, chlorophenols (CP) have potential carcinogenic and mutagenic activity and toxicity. Since there still lacks studies on molecular interactions of chlorophenols with trypsin, one major binding target of many exogenous environmental pollutants, the binding interactions between five chlorophenols, 2-CP, 2,6-DCP, 2,4,6-TCP, 2,4,6-TCP, 2,3,4,6-TCP and PCP and trypsin were characterized by the combination of multispectroscopic techniques and molecular modeling. The chlorophenols bind at the one main site of trypsin and the binding induces the changes of microenvironment and global conformations of trypsin. Different number of chloride atoms significantly affects the binding and the binding constants K-A ranks as K-A (2-CP) < K-A (2,6-DCP) approximate to K-A (2,4,6-TCP) < K-A (2,3,4,6-TCP) < K-A (PCP). These chloro phenols interacts with trypsin mainly through hydrophobic interactions and via hydrogen bonding interactions and aromatic-aromatic pi-pi stacking interaction. Our results offer insights into the binding mechanism of chlorophenols with trypsin and provide important information for possible toxicity risk of chlorophenols to human health. (C) 2014 Elsevier B.V. All rights reserved.