Betaine, also called trimethylglycine, is either produced endogenously by choline oxidation or found naturally in wheat or sugar beets. We hypothesized that betaine supplementation improves the total-tract digestibility and production performance in mid-lactation dairy cows. There were 20 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 yield and feed intake for each cow was recorded daily. During d 21–28, cows were fed with chromic oxide (15 g/d per cow). On d 26–28, fecal samples were collected and analyzed for digestibility via chromic oxide concentration analysis by inductively coupled plasma – atomic emission spectrometry (ICP-AES). Milk samples were collected on d 21 and d 28 for components analysis and fatty acids profile via gas chromatography. Data were analyzed using the Proc Mixed of SAS with significance declared at P ≤ 0.05 and trends at P < 0.1. Apparent total-tract digestibility tended to increase by supplementing 100 g betaine as compared with that with no dietary betaine (0.61 vs 0.58 ± 0.01; P = 0.1). Milk fat percent (3.16 vs 3.36 ± 0.08%, for 0 g betaine/d and 200 g betaine/d, respectively) and C20:1 (0.005 vs 0.004 ± 0.0006%, for 100 g betaine/d and 200 g betaine/d, respectively) tended to differ among treatments (P = 0.1 for both). Results showed however that DMI (25.4, 25.4, 25.4 ± 0.11 kg/d), milk yield (29.7, 29.3, and 30.0 ± 0.7 kg/d), and energy-corrected milk yield (28.5, 28.1, and 29.0 ± 0.8 kg/d) did not differ among treatments (0, 100, and 200g betaine/d per cow, respectively.) Overall, the result showed that betaine supplementation slightly affected the total-tract digestibility and milk fat of mid-lactation dairy cows whereas no major effect was observed in production measures tested.