Aim To identify paclitaxel differential binding protein in the cells of cell line MCF-7 and MCF-7/Taxol and to find new target for antitumor agents.Methods Synthesis and activity assay of biotinylated paclitaxel was used to gain paclitaxel binding protein by semiautomatic in vitro selection using the affinity magnetic beads method.LC MS/MS analysis and Western immunoblot analysis were used to identify the differential binding protein.Results The experimentation identified paclitaxel binding protein in the cells of MCF-7 and MCF-7/Taxol.An absent strap in MCF-7/Taxol was discovered by comparing the straps of two cell lines, which contained 25 kinds of proteins, among which 3 proteins were identified by western blot techniques: Heat shock protein HSP 90,Dermcidin Precursor,Actinin.Conclusions By comparing the straps of two cell lines, the differential protein in the cells of MCF-7 and MCF-7/Taxol are discovered, implying that they may be the novel mechanism of taxane resistance and may lead to find a new approach to finding a new target for oncotherapy drugs.