Objective To construct bone morphogenetic protein 9 (Bmp9)-knockout mice using CRISPR/ Cas9technology. Methods sgRNA were designed and synthesized according to the Bmp9 sequence in Genbank. After in vitrotranscription of Bmp9 sgRNA, a Cas9 and sgRNA mixture was microinjected into zygotes of C57BL/6 mice, which werethen transplanted into ICR mice. The offspring mice were identified with Sanger sequencing and mutant mice were matedwith wild-type mice to screen for mice harboring a homozygous mutation. Meanwhile, qPCR, western blot, andimmunohistochemistry were used to detect the expression of BMP9 in the heart, liver, spleen, lung, and kidney. Results A 20-bp sgRNA was synthesized and transcribed into RNA. After microinjection, transplantation, and mating, homozygousF2-generation mice were obtained. The results of sequencing revealed two genotypes: one with a 5-bp deletion and anotherwith a 13-bp deletion and 1-bp insertion. Compared with the wild-type C57BL/6 mice, results of qPCR, western blot, andimmunohistochemistry showed a marked decrease in BMP9 in the liver of Bmp9-knockout mice. Conclusions Bmp9-knockout mice are successfully generated using CRISPR/ Cas9 technology.