Objective To establish an animal model of calpastatin (CAST) transgenic mice by inserting the full human CAST into the genome of C57BL/6J mice. Methods Recombinant transgenic vector pRP.EX3d-EF1A-CAST-IRES-eGFP was constructed by Gateway technology. It was injected into the fertilized eggs from C57BL/6J mice. The injected eggs were transplanted into the oviduct of pseudopregnant mice. Tail DNA PCR screening was performed to identify the positive founder mice. The expressions of CAST mRNA and protein in tissues of the transgenic mice were detected by RT-PCR and Western blotting. Results Ninty eggs were transplanted into the oviducts of 3 recipients. The transplantation success rate was 100%. 23 viable offsprings were born from the recipients. Tail DNA PCR screening showed that two of the offsprings were positive transgenic mice. The positive rate of transgenic mice was 9%. RT-PCR assay revealed that CAST mRNA expressions were present in the heart, liver, kidney, lung, spleen, brain and skeletal muscle of the transgenic mice. Additionally, the CAST protein expression was significantly increased in the transgenic mice. Conclusion CAST transgenic mice have been successfully established and provide a good animal model support for further studies on the CAST function.