Objective To construct miRNA-29b1 gene knockout mice based on CRISPR/Cas9 technology. Methods To design and synthesize sgRNA according to the miRNA-29b1 sequence in Genbank. sgRNA and Cas9 were transcribed to RNA in vitro, these RNA were then microinjected into zygotes of C57BL/6 mice. After mouse birth, the genome DNA was extracted and sequenced to identify its genotype; meanwhile, real-time PCR was used to assay the expression of miRNA-29b1 in the heart, liver, spleen, lung and kidney of mutated mice. Result A 20 bp sgRNA targeted on miRNA-29b1 was synthesized and transcribed to RNA with Cas9. After microinjection, miRNA-29b1 gene-mutated mice were obtained. The sequencing results showed that there were two types of genotype for the mutated mice, one was 10 bp deletion, and another was 23 bp deletion accompanied with a 3 bp insertion. Compared with the wild-type mice, the expression of miRNA-29b1 in the heart, liver, spleen, lung and kidney was reduced significantly. Conclusions miRNA-29b1 gene knockout mice are constructed successfully by using CRISPR/Cas9 technology.