Bamboo was fractionated using formic acid based on a one-step organosolv process under high pressure (at 145°C for 45min, 0.3MPa) and at atmospheric pressure (at 101°C for 120min), respectively. High-pressure formic acid lignin, atmospheric formic acid lignin and milled wood lignin of bamboo were subjected to comprehensive structural characterization using gel permeation chromatography (GPC), Fourier transform infrared (FTIR) and solution-state NMR techniques (1H, 13C, 2D-HSQC, and 31P-NMR spectroscopies). The results indicated that the formic acid fractionation under high pressure presents a quick and efficient delignification method by enhancing the cleavage of interunitary bonds in lignin (β-O-4′, β-β and β-5′), and dissolved lignin also occurred condensation reaction. As compared to atmospheric formic acid lignin, high-pressure formic acid lignin obtained showed higher purity and yield, and had relatively higher contents of phenolic and carboxylic groups.