In this experiment the response to niacin supplementation was evaluated. Twelve multiparous Holstein cows were used in a replicated 4 × 4 Latin square statistical model in four 24 d periods as replicates. At the onset all the cows were in early lactation with days in milk “EN-GB”>(DIM) at 60.1 ± 16.1 and an average milk yield of 31.4 ± 5.44 L/d. The treatments were: N0 – control (no niacin supplement); N1 – control + 12 g niacin/d; N2 – control + 14 g niacin/d and N3 – control + 16 g niacin/d. Means were compared with the Duncan‘s least range test. Milk yield, fat-corrected milk (FCM, 3.5%) and total solids percentage (TS) were significantly higher in the N2 compared to the other treatments. There were no significant differences between treatments in milk fat and milk lactose percentages, but milk fat yield was significantly higher and milk fat percentage numerically higher in the N2 than in the other treatments. Milk protein yield and percentage were highest in N2, but milk protein percentage was not significantly different between the N2 and N1 treatments. Milk solids non-fat (SNF) percentage was the highest for N2. Evaluation of blood plasma parameters showed that 14 g niacin (N2) supplementation resulted in a significantly higher glucose concentration and the lowest ß-hydroxybutyrate (BHBA) and nonesterified fatty acids (NEFA) concentrations as compared to the other treatments. This study suggested that the optimal level of niacin supplementation was 14 g niacin/cow/d.