The 4-coumarate∶CoA ligase (4CL) is one of the most important enzymes of the plant specific phenylpropanoid pathway. It catalyzes the syntheses of coumaroyl-CoA thioesters, the precursors of lignin and other important phenylpropanoids, from corresponding coumarate compounds such as 4-coumaric acid, caffeic acid, and 3-methoxy-4-coumaric acid, ATP, and coenzyme A, in two-step reactions that involves the formation of coumarate-AMP anhydride as intermediates and subsequent formation of the the coumaroyl-CoA thioesters through the nucleophilic substitution of the AMP group by CoA. Because of the important roles played by 4CL in the biosynthesis of lignin, this protein has become the target of bioengineering aimed at improving the quality of plant products. We have solved the crystal structure of Populus tomentosa 4CL1 (Pt4CL1) complexed with its enzymatic intermediate 4-coumarate-AMP anhydride. The Pt4CL1 was found to adopt the conformation of the second step reaction based on comparisons with other members from the same superfamily. Structural analysis revealed that the residue His-234, which forms a hydrogen bond to the phosphate group of the 4-coumarate-AMP anhydride through its side chain imidazole group, plays essential roles in the enzymatic mechanisms of Pt4CL1, including facilitating both steps of the reactions by forming a hydrogen bond to, and reducing the negative charge of, the phosphate group; de-protonating the thiol group of CoA; and regulating the access of the active site to CoA through the conformational changes of its side chain. Enzymatic assays on Pt4CL1 and relevant mutant verified that His-234 is an essential residue of this enzyme.