Betaine, naturally found in plants and an oxidative product of choline, is converted to acetate in the rumen, which may be used for milk fat synthesis. The objective of this study was to determine the effect of supplemental dietary betaine on milk yield and milk composition. Eighteen Holstein dairy cows (126±5 d in milk; mean ± SD) were randomly assigned to a sequence of treatments of rumen-unprotected betaine at 0, 25, 50, and 100 g/d added to a standard lactation ration in a 4×4 Latin square design. Animals were fed individually with feed intake and milk yield recorded daily. Body condition score and body weight were recorded on the last day of each period that lasted 16 d, with milk sampled on the last 2 d of each period. Milk composition was determined by a Dairy Herd Improvement Association laboratory and milk fatty acids were determined by gas chromatography. Data collected over the last 2 to 3 d were analyzed using the MIXED procedure in SAS (SAS Institute Inc., Cary, NC). Milk yield (mean ± SEM) was increased by betaine when fed at 100g/d (22.4, 22.5, 22.8, 24.1±1.19 kg/d for 0, 25, 50, and 100g of betaine/d, respectively). No effect of dietary betaine was detected on dry matter intake, feed efficiency, body weight, or body condition score. Percentages of milk fat, lactose, solids-not-fat, and somatic cell count were not altered; however, protein concentration was decreased by betaine supplementation as compared with the control (3.35, 3.28, 3.27, and 3.28±0.07% for 0, 25, 50, and 100 g of betaine/d, respectively). Daily yields of milk protein, fat, lactose, energy-corrected milk, and 3.5% fat-corrected milk did not differ with betaine supplementation. Overall, inclusion of dietary betaine at 100 g/d increased milk yield, whereas all levels of betaine supplementation decreased milk protein percent and slightly altered milk fatty acid profile. Further studies are needed to determine the ruminal fermentation characteristics and the optimum rate of supplemental betaine for dairy cows.