Very long chain polyunsaturated fatty acids (VLC-PUFAs) are unique fatty acids in tissues of mammals such as retina and testis, and the key enzyme of its biosynthesis is very long chain fatty acid elongase 4 (Elovl4). Development of an animal model of tissue-specific knockout of Elovl4 gene is conducive to the in-depth study of the biological function of VLC-PUFAs. Therefore, we constructed Stra8-Cre mice and Elovl4 floxed mice based on Cre/loxP system, and obtained the (Elovl4[flox/+], Stra8-Cre) heterozygous knockout mice by hybridization. Subsequently, female mice were selected to cross with male mice with homozygous Elovl4[flox/flox] to gain homozygous mice (Elovl4[flox/flox], Stra8-Cre) through genotype identification and screening. RT-PCR, qRT-PCR, Western blotting, immunohistochemistry and immunofluorescence techniques were used to detect the knock-out efficiency of Elovl4 in testis. The expression of Elovl4 in testis of both heterozygous and homozygous knockout mice were significantly down-regulated at mRNA and protein levels, but were not affected in other tissues. In summary, we constructed a mouse model with specific knockout of Elovl4 gene in testis, which provides a reliable animal model for studying the effect of VLC-PUFAs on the reproductive function of male mice and the underpinning molecular mechanisms.