p -Coumaroyl-CoA 3′-hydroxylase (C3′H) is a cytochrome P450-dependent monooxygenase that catalyzes the 3′-hydroxylation of p -coumaroyl shikimate and p -coumaroyl quinate. We used RNA interference to generate transgenic hybrid poplar suppressed in C3′H expression and analyzed them with respect to transcript abundance, cell wall structure and chemical composition, and soluble metabolite levels. RT-PCR expression profiles confirmed the down-regulation of C3′H in a number of lines, which generally correlated very well with reduced total cell wall lignin content. The most strongly repressed line was chosen for further analysis and compared with the wild-type trees. In-depth characterization revealed that along with the significant decrease in total lignin content, a significant shift in lignin monomer composition was observed, favoring the generation of p -hydroxyphenyl units at the expense of guaiacyl units while the proportion of syringyl moieties remained constant. Suppression of C3′H also resulted in the accumulation of substantial pools of 1- O-p -coumaroyl-β- d -glucoside and other phenylpropanoid glycosides, and p -coumaroyl shikimate, providing further insight into the role of C3′H in the lignin biosynthetic pathway. The data presented indicate that when down-regulated, C3′H becomes a rate-limiting step in lignin biosynthesis and further support the involvement of hydroxycinnamic acid shikimate esters in the lignin biosynthetic pathway.