Objective：To evaluate the relationship between the nuclear factor-κB（NF-κB）family member RelB and tumor metastasis，we established a RelB knockout mouse model by CRISPR/Cas9 system. Methods：The RelB specific single-guide RNAs targeted exon4 were designed for CRISPR/Cas9 system. The Cas9 and sgRNAs were transcribed by T7 RNA polymerase and microinjected into the mouse zygote. C57BL/6 mouse fertilized eggs injected with sgRNA and Cas9 mRNA in vitro were transplanted into mouse uterus. F1 generation was obtained by mating F0 generation with wild type. PCR and gene sequencing were performed to identify the RelB phenotype of F0 and F1 generation mice. The expression of RelB in F0 and F1 generation mice was evaluated in gene level and protein level. Results：We obtained four RelB-/+ F0 mice and stable inherited RelB-/- offspring. The results of genome sequencing showed that there was a nonsense mutation in the mRNA of RelB in the knockout group，which caused the mRNA translation to terminate. Compared with the control group，the expression of Relb protein was not detected in the knockout group. Meanwhile，the results of Western blot showed that there was no significant difference between knockout group and control group in the expression of NF-κB family members（RelA，p50 and p52）. Conclusion：The RelB knockout C57BL/6 mouse model was successfully established，which provided an important tool for the study of RelB in tumor metastasis and drug resistance.