Sixteen multiparous lactating Holstein cows were used in 2 experiments to evaluate the effects of reduced-fat dried distillers grains with solubles (RFDG) on milk production, rumen fermentation, intestinal microbial N flow, and total-tract nutrient digestibility. In experiment 1, RFDG was fed at 0, 10, 20, or 30% of diet dry matter (DM) to 12 noncannulated Holstein cows (mean ± standard deviation: 89±11d in milk and 674±68.2kg of body weight) to determine effects on milk production. In experiment 2, the same diets were fed to 4 ruminally and duodenally cannulated Holstein cows (mean ± standard deviation: 112±41d in milk; 590±61.14kg of body weight) to evaluate the effects on rumen fermentation, intestinal flow of microbial N, and total-tract nutrient digestibility. In both experiments, cows were randomly assigned to 4×4 Latin squares over 21-d periods. Treatments (DM basis) were (1) control (0% RFDG), (2) 10% RFDG, (3) 20% RFDG, and (4) 30% RFDG. Feed intake and milk yield were recorded daily. In both experiments, milk samples were collected on d 19 to 21 of each period for analysis of milk components. In experiment 2, ruminal pH was measured; samples of rumen fluid, duodenal digesta, and feces were collected on d 18 to 21. Microbial N was estimated by using purines and DNA as microbial markers. Milk yield was not affected by treatment and averaged 34.0±1.29kg/d and 31.4±2.81kg/d in experiments 1 and 2, respectively. Percentage of milk protein tended to increase in experiment 1; estimates were 3.08, 3.18, 3.15, and 3.19±0.06% when RFDG increased from 0 to 30% in the diets. However, milk protein concentration was not affected in experiment 2 and averaged 3.02±0.07%. Percentage of milk fat was not affected and averaged 3.66±0.05% and 3.25±0.14% in experiments 1 and 2, respectively. Total ruminal volatile fatty acids and ammonia concentrations were not affected by treatment and averaged 135.18±6.45mM and 18.66±2.32mg/dL, respectively. Intestinal microbial N flow was not affected by treatment; however, purines yielded higher estimates of flow compared with DNA markers. When averaged across treatments, intestinal flow of microbial N was 303 and 218±18g of N/d, using purines and DNA as the markers. Dry matter, organic matter, neutral detergent fiber, and nonfiber carbohydrate digestibility tended to increase with increasing inclusion of RFDG. Results from these experiments indicate that dairy rations can be formulated to include up to 30% RFDG while maintaining lactation performance, volatile fatty acids concentration, and intestinal supply of microbial N.