Cyclin M2(CNNM2) is one of the regulatory transport genes involved in zebrafish Mg²⁺ homeostasis. We generated CNNM2 gene knockout zebrafish by CRISPR/Cas9 system which has been developed into a novel gene targeting system in recent years.First, we constructed the sgRNA expression vector targeting zebrafish CNNM2 gene. Then, after in vitro transcription of sgRNA and Cas9 vectors, we co-injected them into one-cell fertilized eggs of zebrafish to generate zebrafish with targeted mutation. After 24-48 h, injected embryos were collected and used to conduct mutation analysis with restriction endonuclease and sequencing. To further verify the stability of genetic mutations, we selected mutated founder to outcross with wild type zebrafish. A knockout and wild-type outcross F₀ efficiency were obtained, and then F₁ were analyzed by PCR and sequencing of the caudal fin. The results showed F₀ and F₁ were introduced insertion, deletion and frameshift mutations to some extent, and F₀ mutation efficiency was 88%. The CNNM2 gene knockout zebrafish generated in this study can be transmitted by germline and provide a basis for further study of CNNM2 and Mg²⁺ homeostasis.