The CCCTC-binding factor (CTCF) is the main insulator protein described in vertebrates. It plays fundamental roles during diverse cellular processes. CTCF gene knockout mice led to death during embryonic development. To further explore the functions of CTCF, we employed a CRISPR/Cas9-based genome engineering strategy to in-frame insert the mitosis-special degradation domain (MD) of cyclin B into the upstream open reading frame of CTCF gene. Fusion protein is designed to degrade during mitosis leaded by MD. As a control group, mutation of a single arginine (R42A) within the destruction box inactivates the MD leading to constitutive expression of MD*-CTCF. The homozygous clones were obtained via the screening by puromycin when coexpressed with puromycin resistence gene. The protein level of CTCF in MD-CTCF cell line was about 10% of wild-type cells throughout cell cycles by the analyses of Western blotting and immunofluorescence. There was no significant difference between MD*-CTCF cell line and wild type. Flow cytometry results showed prolonged G1 phase in MD-CTCF cell line. Taken together, we demonstrated the feasibility of efficiently inserting MD domain into genome with the CRISPR/Cas9 technology and reported the first CTCF-specific degradation human cell line.