Purpose <p>Vinasse (VIN), a by-product of sugarcane ethanol or sugar production, is a nutrient-rich liquid with potential as a feed ingredient for ruminants and enhance dietary efficiency and sustainability. This experiment was conducted to assess the influence of substituting dietary urea with VIN on dry matter (DM) intake (DMI), lactating performance, nutrient digestibility and some biochemical blood metabolites in dairy cows.</p> Methods <p>Fourty Holstein dairy cows (185 ± 21 d in milk, 35 ± 3.6&#xa0;kg of milk/d, 670 ± 31&#xa0;kg body weight) were randomly selected and divided into two groups (<i>n</i> = 20 per treatment) in a complete randomized design. The study comprised two experimental groups: a control group (V0) receiving a diet containing 0.2% urea on a DM basis, and a treatment group (V2) supplemented with 2% VIN. Milk yield and composition parameters including fat percentage, true protein, lactose, solids not-fat (SNF) and milk urea nitrogen (MUN) were determined. Blood samples were taken at 40 d and analyzed for glucose, cholesterol, triglycerides, albumin, globulin, total protein and blood urea nitrogen (BUN).</p> Results <p>The results indicated that there was a trend toward increased DMI (<i>P</i> = 0.09), milk yield (<i>P</i> = 0.06), and kg of fat yield (kg/d; <i>P</i> = 0.06) in cows fed VIN compared to the urea-based control diet. Significant improvements (<i>P</i> = 0.01) were observed in fat-corrected milk (FCM), energy-corrected milk (ECM), lactose yield (kg/d), and SNF yield for VIN-fed cows relative to control. However, milk composition parameters including fat percentage (<i>P</i> = 0.74), true protein (<i>P</i> = 0.11), lactose (<i>P</i> = 0.88), SNF percentage (<i>P</i> = 0.17) and MUN (<i>P</i> = 0.50) remained unaffected by dietary treatments. Compared to the urea-based control diet, VIN supplementation significantly improved the digestibility of DM (<i>P</i> = 0.03), organic matter (OM; <i>P</i> = 0.02) and neutral detergent fiber (NDF; <i>P</i> = 0.02) while, crude protein, acid detergent fiber and ether extract digestibility did not differ (<i>P</i> &gt; 0.02) between treatments. Blood metabolite concentrations, including glucose, cholesterol, triglycerides, albumin, globulin, total protein and BUN remained unaffected (<i>P</i> &gt; 0.05) by dietary treatments.</p> Conclusion <p>Overall, partial replacement of urea (0.2% of dietary DM) with VIN (2% of dietary DM) in diet of mid-lactation dairy cows enhanced FCM, ECM, lactose and SNF yield, improved DM, OM and NDF, and showed a tendency to increase DMI.</p>