The effects of corn dried distillers grains with solubles (DDGS) feeding on rumen fermentation and milk production in cows were evaluated using diets high in neutral detergent fiber (NDF, 45.9–46.6%). The control diet (Control) consisted mainly of hay, corn silage and concentrates. In the experimental diets, the concentrates were replaced with DDGS as 10% dry matter (DM) (10%DDGS) and 20% DM (20%DDGS). Eight cows were used for each 14-day treatment period. Effect of DDGS feeding on DM intake was not significant. Ruminal volatile fatty acids and ammonia-N at 5 h after feeding of 20%DDGS were decreased compared to Control, whereas protozoal count at 2 h after feeding of 20%DDGS was higher than that of 10%DDGS. Milk yield of cows fed DDGS diets was greater than that of Control, although percentages of milk protein and solids-not-fat were decreased by DDGS diets. The proportions of C10:0, C12:0, C14:0 and C16:0 in the milk fat decreased, and those of C18:0, C18:1, C18:2 and cis-9, trans-11 conjugated linoleic acid (CLA) increased markedly with elevated DDGS. Increase in trans-11 C18:1 was observed in the rumen fluid at 5 h after feeding. These findings suggest that DDGS feeding enhanced milk yield, as well as CLA synthesis under a high dietary NDF condition.