A novel label-free quantitative proteomic platform of SWATH-MS was applied to detect proteome changes in poplar seedling roots following Pb treatment. In total 4388 unique proteins were identified and quantified, among which 542 proteins showed significant abundance changes upon Pb(II) exposure. The analysis of these differentially expressed proteins revealed significant proteome changes in lignin and flavonoid biosynthesis, cell wall metabolism as well as glutathione metabolism. Furthermore, a certain proportion of proteins involving RNA processing serve as a regulatory role in poplar Pb(II) detoxification. This work represents the first SWATH-MS analysis in model woody plant poplar and highlights mediating roles of potential aspects in poplar during Pb stress, which would provide new cues for the treatment of heavy metal contaminated-soil and would extend our strategies on future environment protection.