Dichlorvos (DDVP) is widely applied in the agricultural industry, and its residues are considered hazardous to the environment. Microbial bioremediation is an innovative technology with the potential to mitigate such pollution. Trichoderma atroviride strain T23, a filamentous fungus, is very efficient at degrading DDVP. Therefore, we used DDVP as a model organophosphate pesticide to study the mechanism by which Trichoderma degrades organophosphate pesticides, with the aim of attaining a global understanding of the molecular mechanism of enzymatic degradation of organophosphate pesticides by beneficial fungi. DDVP can be biodegraded via two routes, and the primary one involves hydrolysis of the P-O bond, which can result in the production of the novel degradation intermediate trichloroethanol. TaPon1-like showed continuously high expression during 120 h, and deletion of the gene decreased the efficiency of P-O bond hydrolysis. The enzyme produced by TaPon1-like had a low K-m for DDVP (0.23 mM) and a high k(cat) (204.3 s(-1)). The enzyme was able to hydrolyze broad substrates such as organophosphate oxons and lactone and maintain stable activity in a wide range of pH and temperature values. The TaPon1-like hydrolase played an important role in the first step of DDVP degradation by strain T23 and contributed to a comprehensive understanding of the mechanism of organophosphate pesticide degradation.