Based on the online bioinformatics analysis and alignment results, three RNAi sequences target to the Mus musculus Bicc1 gene were obtained. The three interference fragments were synthesized and cloned into pRS-Hush shRNA Vector. The Bicc1 eukaryotic expression vector pEGFP-C3-Bicc1 was constructed, tagging the GFP to the N-terminal of the Bicc1 protein. The pEGFP-C3-Bicc1 and three pRS-Hush-RNAi were co-transfected into the cultured HEK-293 cells line, respectively. The two RNAi (pRS-Hush-RNAi-Bicc1-N/-C) that could knock-down the Bicc1 expression levels in HEK-293 cells significantly were confirmed by cell immunofluorescent staining, semi-quantitative PCR and Western blotting. The results demonstrate that we have successfully obtained two efficent Bicc1 RNAi sequences, which lays a foundation for further studying on the construction of Bicc1 knock-down stable cell lines and biological function of mouse Bicc1 product.