Omega-3 polyunsaturated fatty acids (n-3 PUFAs) are beneficial to human health. However, the n-3 PUFAs contents of the livestock meat that we consume daily are relatively low. Utilization of transgenic technology to increase n-3 PUFAs contents in livestock may solve this problem. The omega-3 fatty acid desaturase (FAD3), encoded by fat1 gene derived from Caenorhabditis elegans (C. elegans), converts omega-6 polyunsaturated fatty acids (n-6 PUFAs) to n-3 PUFAs. In the study, a plasmid containing the codon-optimized C. elegans fat1 gene (mfat1) was constructed and used to produce transgenic beef cattle by somatic cell nuclear transfer. Fourteen transgenic calves were obtained, and the ratio of n-6 to n-3 PUFAs in the transgenic calves decreased from 5.33: 1 to 0.95: 1 compared with negative controls. Our results demonstrated that the codon-optimized C. elegans mfat1 gene can be functionally expressed in the beef cattle and converts n-6 PUFAs to n-3 PUFAs.