Objective This study aimed to investigate method for the optimal breeding and identification of vitamin D receptor (VDR) knockout mice, which could provide animal models for further research on VDR function and related diseases. Methods Based on VDR knockout mice previously obtained in our lab, four different mating methods were used to generate offsprings, the phenotypes and genotypes of which were evaluated and the reproductive ability of VDR knockout homozygous (VDR- / - ) and heterozygous (VDR+ / - ) mice were compared. And, VDR expression was analyzed at the mRNA and protein levels. Results Compared with VDR+ / + mice, VDR- / - mice had a significantly different phenotype, mainly in terms of having less hair, small size, and decreased mobility. The genotype distributions of the offspring mice produced by the four breeding method were basically in accordance with Mendel’ s law of inheritance.VDR- / - cross-over had poor reproductive ability and could not be directly used to expand VDR- / - homozygous knockout mouse strains, while VDR+ / - cross-over could successfully obtain VDR- / - mice. It was also determined that VDR knockout traits could be stably inherited by offspring mice, so VDR+ / - cross-over is the best way to expand the strains.Conclusions This experiment has successfully identified the genotypes of offspring mice and established VDR knockout mouse strains, laying the foundation for subsequent research on VDR function.