The objective was to investigate the regulatory effect of polyunsaturated fatty acids (PUFAs) on mRNA expression of key genes involved in homocysteine (Hcy) metabolism. Eighty male Sprague Dawley rats were randomly divided into eight groups. The oils were orally administered daily for 8 weeks. Plasma Hcy, phospholipids fatty acids, and mRNA expression were determined. Compared with the control group, plasma Hcy was significantly decreased in the 22:6n-3and conjugated linoleic acid (CLA) groups; mRNA expression ofMthfrwas significantly upregulated in the 22:6n-3, 20:5n-3, and 18:3n-3groups and downregulated in the 18:2n-6and stearolic acid (SO) groups.Mat1awas upregulated in the 22:6n-3, 20:5n-3, 18:3n-3, and CLA groups. In addition,Cbswas upregulated in the 22:6n-3, 20:5n-3, 18:3n-3and CLA groups while downregulated in 18:2n-6and SO groups. Dietary 22:6n-3and CLA decrease the plasma concentration of Hcy. mRNA expression ofMthfr, Mat1a, CbsandPemt, Gnmt, Mtrr, andBadis upregulated byn-3PUFA and downregulated byn-6PUFA. CLA upregulates mRNA expression ofMat1aandCbs.