This study was undertaken to investigate the effects of dicarboxylic acid supplementation, as replacement antibiotics, of on in vitro ruminal parameters and milk yield and milk composition in lactating cows. in vitro treatments were 1) Con (4 g of basal diet), 2) CM (4 g of basal diet + 0.05 ml of monensin), 3) CR (4 g of basal diet + 0.1 ml of dicarboxylic acid) and in vivo treatments were 1) Con (25 kg of basal diet/head/day), and 2) CR (25 kg of basal diet + 5 g of dicarboxylic acid/head/day), respectively. A total 10 lactating dairy cows (649±19 kg average body weight, 99±65 average milking days) were divided in to two groups according to mean milk yield and number of days of postpartum. The cows fed a basal diet during adaptation (2 wk) and experimental diets during the treatment periods (4 wk). In the first in vitro experiment, there were no statistical differences between treatments in pH, gas production, and ammonia-N and lactic acid concentration during incubation. However, dry matter digestibility was significantly higher in CR treatment compared to control or CM treatment (P<0.05). Total VFA was tended to higher in CR treatment than those of control and CM treatment (P> 0.05). In the second experiment, milk yield was significantly higher in treatment (40.39 kg) compared to control (35.19 kg), (P<0.05). Milk composition and MUN were not changed by dietary supplementing dicarboxylic acid. Therefore the present results reporting that supplementation of dicarboxylic acid might enhance the stabilization of ruminal fermentation and increase the milk yield of lactating cows.