Abstract: This study is to explore new lead compounds by inhibition of Pin1 for anticancer therapy using temperature sensitive mutants.As Pin1 is conserved from yeast to human, we established a high-throughput screening method for Pin1 inhibitors, which employed yeast assay.This method led to the identification of one potent hits, 8-11.In vitro, 8-11 inhibited purified Pin1 enzyme activity with IC₅₀ of (10.40 ± 1.68) μmol·L^-1, induced G₁ phase arrest and apoptosis, showed inhibitory effects on a series of cancer cell proliferation, reduced Cyclin D1 expression, was defined as reciprocally matched for protein-ligand complex in virtual docking analysis and reduced cell migration ability.In vivo, we could observe reduction of tumor volume after treatment with 8-11 in xenograft mice compared with vehicle DMSO treatment.Altogether, these results provide for the first time the involvement of 8-11 in the anticancer activity against Pin1.