n-3 Polyunsaturated fatty acids (n-3 PUFAs) contribute to preventing many types of diseases, including cancer; however, a high n-6 polyunsaturated fatty acids (n-6 PUFAs) intake in modern diets has the opposite effect. Previously, we developed a transgenic mouse model that expresses a gene, fat-1, encoding an n-3 fatty acid desaturase, which converts n-6 PUFAs to n-3 PUFAs in vivo. MicroRNAs (miRNAs) in serum are stable, reproducible, and consistent among individuals of the same species and serve as potential biomarkers for the detection of cancers and other diseases. Employing illumina sequencing, we analyzed all the serum miRNAs in wild-type and mfat-1 transgenic mice. Using quantitative real-time PCR (RT-qPCR), we identified 12 miRNAs that were highly expressed in mfat-1 mice. Pathway analysis of targets regulated by these miRNAs revealed a significant number of genes involved in the development of cancer, including phosphatidylinositol 3-kinase (PI3K), mitogen-activated protein kinases (MAPK), and mammalian target of rapamycin (mTOR), which suggested a relationship between n-3 PUFAs and cancer prevention.