In the present study, the plantlets in vitro of Paeonia suffruticosa ‘Wu Long Peng Sheng’ were used as laboratory materials. The proteome during adventitious root induction process was investigated to sift the related proteins by two-dimensional electrophoresis and mass spectrometry. The results indicated that the protein spots were concentrated in the acidity gel region (pH 4 - 7) and the spots number had a dynamic change ranged from 373 to 462 at the process of root induction (0 – 7 d). 8 spots significantly changed were analyzed with a mass spectrometer and identified using associated software and databases. The peptide information of the 8 spots was similar to the ATP synthase β-subunit of P. suffruticosa (Spots 1 - 4 and 8), P. tenuifolia (Spots 5), P. californica (Spot 6) and P. brownie ( Spots 7) r espectiv ely. T he expression levels of protein spots 1, 4, 5, 6 and 7 was dramatically downregulated, and that of protein spots 2 and 3 had a slightly opposite tendency on the 3rd day. The obviously decreased period is particularly interesting as it was consistent with the induction period of adventitious root primordial of tree peony plantlet in vitro. The ATP synthase β-subunit could be consumed for assembling the ATP synthase in order to supply energy to the rooting process. Therefore, we speculated that the ATP synthase β-subunit was involved in adventitious root initiation of tree peony plantlets in vitro and we expect that further studies should be carried out in order to export its action mechanism.