The objective of the present study was to evaluate the effects of betaine supplementation on rumen fermentation, lactation performance and plasma characteristics in dairy cows. Twenty multiparous Holstein dairy cows (597±11·8 kg body weight (BW), 88±4·5 days in milk (DIM) and average daily milk production of 26·3±0·5 kg/cow) were used in a replicated 4×4 Latin square experiment. The treatments were: control (without betaine), low-betaine (LB), medium-betaine (MB) and high-betaine (HB) with 0, 50, 100 and 150 g supplemental anhydrous betaine/cow/day, respectively. Betaine was hand-mixed into the top one-third of the daily ration at feeding. Experimental periods were 30 days with 15 days of adaptation and 15 days of sampling. Dry matter (DM) intake was not affected with increasing the betaine supplementation. There were linear increases in milk yield and fat-corrected milk yield (corrected to 40 g fat/kg) and a linear and quadratic increase in milk fat concentration with increasing the betaine supplementation, whereas the proportion and yield of milk protein and lactose, and feed efficiency, were not affected. Ruminal pH and ammonia N linearly decreased, whereas total volatile fatty acid (VFA) concentration linearly and quadratically increased with increasing the betaine supplementation. The ratio of acetate to propionate (A:P) linearly increased from 3·06 to 3·53 as betaine supplementation increased. Digestibility of DM linearly increased, whereas digestibilities of organic matter (OM), crude protein (CP), neutral detergent fibre (NDF) and acid detergent fibre (ADF) in the total tract were quadratically increased with increasing the betaine supplementation. Plasma concentrations of non-esterified fatty acids (NEFA) and β-hydroxybutyrate (BHBA) were lower for betaine supplementation than for control, and were linearly decreased by betaine supplementation. The results indicate that supplementation of mid-lactation dairy cow diets with betaine increased milk yield through increased feed digestion. Betaine supplementation may benefit lactation performance when methionine supply is limiting.