Mechanical bending treatment was employed to cause tension wood formation in the stem of the fast-growing yellow poplar. The tension wood induced by mechanical bending had many characteristic features of tension wood, i.e., eccentric growth toward tension wood, increased frequency of fiber cell types, vessels with reduced size, and reduced lignin content in the developed xylem. The significant reduction of the guaiacyl (G) and p-hydroxyphenyl (H) units of lignin in the tension wood was clearly visualized in samples that had been subjected to bending for 7 and 14 days when analyzed by lignin histochemical analysis using phloroglucinol-HCl. The syringyl (S) unit in the tension wood lignin was also significantly decreased in the developed xylem of the 14-day bending treatment sample, as indicated by Maule’s staining. Expression analysis of several representative genes in the lignin biosynthetic pathway clearly demonstrated that the overall phenylpropanoid pathway toward biosynthesis for both the lignin monomers and the flavonoids was greatly downregulated on bending treatment. In addition, the genes encoding laccases, which have been reported to be involved in the polymerization of monolignols to produce lignin macromolecules, were significantly downregulated in the tension wood. Despite the very limited sequence information for the genes/enzymes in the phenylpropanoid pathway in yellow poplar, histochemical staining and expression analysis using quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) successfully demonstrated the anatomical and chemical characteristics associated with mechanical bending-induced tension wood formation in the yellow poplar.