Lignin biosynthesis is an essential physiological activity of vascular plants if they are to survive under various environmental stresses on land. The biosynthesis of lignin proceeds in the cell wall by polymerization of precursors; the initial step of lignin polymerization is the transportation of lignin monomers from the cytosol to the cell wall, which is critical for lignin formation. There has been much debate on the transported form of the lignin precursor, either as free monolignols or their glucosides. In this study, we performed biochemical analyses to characterize the membrane transport mechanism of lignin precursors using angiosperms, hybrid poplar (Populus sieboldii × Populus grandidentata) and poplar (Populus sieboldii), as well gymnosperms, Japanese cypress (Chamaecyparis obtusa) and pine (Pinus densiflora). Membrane vesicles prepared from differentiating xylem tissues showed clear ATP-dependent transport activity of coniferin, whereas less than 4% of the coniferin transport activity was seen for coniferyl alcohol. Bafilomycin A1 and proton gradient erasers markedly inhibited coniferin transport in hybrid poplar membrane vesicles; in contrast, vanadate had no effect. Cis-inhibition experiments suggested that this transport activity was specific for coniferin. Membrane fractionation of hybrid poplar microsomes demonstrated that transport activity was localized to the tonoplast- and endomembrane-rich fraction. Differentiating xylem of Japanese cypress exhibited almost identical transport properties, suggesting the involvement of a common endomembrane-associated proton/coniferin antiport mechanism in the lignifying tissues of woody plants, both angiosperms and gymnosperms.