The use of ionic liquid (IL) in biomass pretreatment has received considerable attention recently because of its effectiveness in decreasing biomass recalcitrance to subsequent enzymatic hydrolysis. To understand the structural changes of lignin after pretreatment and enzymatic hydrolysis process, ionic liquid lignin (ILL) and subsequent residual lignin (RL) were sequentially isolated from ball-milled birch wood. The quantitative structural features of ILL and RL were compared with the corresponding cellulolytic enzyme lignin (CEL) by nondestructive techniques (e.g., FTIR, GPC, quantitative (13)C, 2D and (31)P NMR). The IL pretreatment caused structural modifications of lignin (cleavage of β-O-4 ether linkages and formation of condensed structures). In addition, lignin fragments with lower S/G ratios were initially extracted, whereas the subsequently extracted lignin is rich in syringyl unit. Moreover, the maximum decomposition temperature (T(M)) was increased in the order ILL < RL < CEL, which was related to the corresponding β-O-4 ether linkage content and molecular weight (M(w)). On the basis of the results observed, a possible separation mechanism of IL lignin was proposed.