Betaine is a product of choline oxidation in the body and an ingredient of wheat and sugar beets. Betaine can donate one methyl group to transfer homocysteine into methionine, which is involved in the phosphatidylethanolamine N-methyltransferase (PEMT) pathway. We hypothesized that betaine supplementation affects the serum fatty acids (FA) profile in mid-lactation dairy cows. There were 21 mid-lactation dairy cows assigned to a 3 × 3 Latin square design with 3 periods of 28 d each and 3 treatments of betaine (0, 100, and 200 g/d). Milk samples collected on d 21 and d 28 and blood samples obtained on d 26 to 28 of each period were used for FA analysis via gas chromatography with flame ionization detector and an Agilent HP-88 column (100 m × 0.25 mm with 0.2-μm film thickness, Agilent Technologies). Individual FA was identified by comparison to the standard mixture Supelco 37 FAME (Supelco, Bellefonte, PA). Data were analyzed using the Proc Mixed of SAS with significance declared at P ≤ 0.05 and trends at P ≤ 0.1. Results showed that no change was observed in the content of total serum saturated FA (40.5, 40.4, and 40.3 ± 0.6% for 0, 100, and 200 g betaine, respectively; P = 0.96). The total serum monounsaturated FA decreased with betaine supplementation (16.2, 15.2, and 14.9 ± 0.32%, for 0, 100, and 200 g betaine, respectively; P = 0.01). Serum FA profile showed a decline in the n-6 to n-3 ratio (6.80, 7.07, and 6.50 ± 0.16%, for 0, 100, and 200 g betaine, respectively; P = 0.04). Results showed however, that milk FA profile did not differ among treatments (0, 100, and 200g betaine/d per cow, respectively). Overall, our study demonstrated that betaine supplementation affected the total serum FA profile in mid-lactation dairy cows without affecting the milk FA profile.