Lignin engineering is a promising tool to reduce the energy input and the need of chemical pre‐treatments for the efficient conversion of plant biomass into fermentable sugars for downstream applications. At the same time, lignin engineering can offer new insight into the structure–function relationships of plant cell walls by combined mechanical, structural and chemical analyses. Here, this comprehensive approach was applied to poplar trees (Populus tremula × Populus alba) downregulated for CINNAMYL ALCOHOL DEHYDROGENASE (CAD) in order to gain insight into the impact of lignin reduction on mechanical properties. The downregulation of CAD resulted in a significant decrease in both elastic modulus and yield stress. As wood density and cellulose microfibril angle (MFA) did not show any significant differences between the wild type and the transgenic lines, these structural features could be excluded as influencing factors. Fourier transform infrared spectroscopy (FTIR) and Raman imaging were performed to elucidate changes in the chemical composition directly on the mechanically tested tissue sections. Lignin content was identified as a mechanically relevant factor, as a correlation with a coefficient of determination (r²) of 0.65 between lignin absorbance (as an indicator of lignin content) and tensile stiffness was found. A comparison of the present results with those of previous investigations shows that the mechanical impact of lignin alteration under tensile stress depends on certain structural conditions, such as a high cellulose MFA, which emphasizes the complex relationship between the chemistry and mechanical properties in plant cell walls.