To establish a conditional knocking down system by RNA interfering, the cassette of LoxP-neo-LoxP was inserted into the pBS/U6/CHIPi construct. The destination vector is named pLoxP/CHIPi, which generates an siRNA targeting CHIP (C-terminal hsc70 interacting protein) gene, dependent on expression of the CRE recombinase. In this conditional knocking down model, it is demonstrated that the siRNA of CHIP could effectively decrease not only the mRNA level but also the exogenous or endogenous protein level in mammalian cells after CRE recombinase was expressed, as testing by RT-PCR and Western blot analysis. Luciferase assay showed that transfection of pLoxP/CHIPi with Cre released the CHIP-mediated inhibition of TGF-β signaling. Significantly, pLoxP/CHIPi facilitated the TGF-β signal transduction in the presence of CRE. These results demonstrate that the conditional knocking down system was successfully constructed, and was applicable for further investigation of the negatively regulatory effect of CHIP on TGF-βsignal pathway. The study also provide a powerful tool for further study on the molecular mechanisms and path-physiologic basis related to CHIP-mediated TGF-β signal pathway.