The aromatic nature of lignin makes it a potential renewable source of chemicals and other valuable products. Isolation of lignin from lignocellulosic biomass using organic solvents enables the production of high-purity lignin. The use of formic acid in the organosolv pulping and fractionation process has been widely studied. Characterization of lignin is necessary to achieve valueadded applications of lignin. To simplify the isolation of formic acid-treated lignin, herein, milled wheat straw lignin (MWSL) was employed as an archetype for characterization of the structural changes of lignin during formic acid treatment. The results showed that the MWSL was GSH-type (comprising p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) monolignols) and underwent structural changes during formic acid treatment. Lignin was esterified during the formic acid treatment. The content of alkyl hydroxyl groups in lignin decreased upon formic acid treatment, corresponding to an increase of the number of double bond equivalents (DBE). Lignin units with active reaction sites were liable to slight condensation, which resulted in a moderate increase of the molecular weight. The molecular weight distribution of formic acid-treated MWSL (FMWSL) was wider than that of the MWSL, although the molecular weight of both species did not differ significantly. The β-O-4 linkage in lignin was partially cleaved during formic acid treatment, resulting in the production of new phenolic structures. This improved the solubility of lignin in the cooking liquor and its reactivity for downstream applications.