The objective was to evaluate the performance and milk composition of cows fed diets containing dried distillers' grains with solubles (DDGS). Five multiparous Holstein cows were distributed in a 5 × 5 Latin square. The treatments involved inclusion levels of 0%, 8%, 16%, 24%, and 32% of DDGS from corn in the diet. The inclusion of DDGS in the diet reduced the intake of dry matter (DM), crude protein (CP), and non-fibrous carbohydrates (NFC), and increased the intake of ether extract (EE) and neutral detergent fiber (NDF) (P < 0.05). However, the energy intake was unchanged, which can be explained by the increased digestibility of nutrients. There was a quadratic effect (P < 0.05) on digestibility of DM and NFC, and NDF digestibility increased linearly with the inclusion of DDGS in the diet. Possibly, the reduction of ingested DM promoted a decrease in the passage rate of feed through the gastrointestinal tract resulting in increased digestion. Ruminal microbial protein synthesis was not influenced by the treatments. The inclusion of DDGS promoted a linear increase in the concentration of cholesterol before feeding and in the concentrations of cholesterol and triglycerides four hours after feeding (P < 0.05). Milk production, energy corrected milk, protein content and milk urea nitrogen concentration were unchanged with the inclusion of DDGS in the diet. However, milk fat decreased linearly (P < 0.05) probably due to the high concentration of polyunsaturated fatty acids present in DDGS, that after incomplete biohydrogenation in the rumen form fatty acids responsible for the depression of milk fat. Thus, the inclusion of 32% DDGS from corn in the diet did not alter the performance of lactating cows, however, it reduced milk fat content. All rights reserved, Elsevier.