AbstractDetermining the structural changes of lignin during bio-treatment will facilitate the understanding of biomass recalcitrance during the sustainable production of chemicals and fuels. However, the analysis of milled wood lignin (MWL) cannot completely elucidate the complex and irregular structural changes therein. In this study, MWL and lignin degradation compounds were extracted from white-rot fungi-treated poplar in order to unveil the degradation process. Results from MWL revealed that the cleavage of β-O-4′ linkages (from 76.4/100Ar to 31.5/100Ar) and the degradation of β–β′and β-5′ linkages clearly occurred, resulting in a decrease in molecular weight. In addition, G-type lignin was more degraded than S-type lignin, with a slightly elevated S/G ratio from 1.13 to 1.29. Further analyses of lignin degradation compounds confirmed these results by showing a high amount of conjugated and unconjugated C=O functionalities. Furthermore, the degradation product of G-type lignin (vanillin) was detected by 2D HSQC NMR and GC-MS. This study of lignin alterations during white-rot fungi treatment could be beneficial for the sustainable production of chemicals, materials, and fuels from renewable plant resources.