The objective of Trial 1 was to evaluate in dairy cows the effects of dietary supplementation with biotin and a B-vitamin blend on dry matter intake (DMI), milk yield, composition and component yields, total tract nutrient digestion, and plasma metabolites. Twenty-four multiparous Holstein cows averaging 46 +/- 8 d in milk at trial initiation were randomly assigned to treatments in a replicated 4 x 4 Latin square design with 28 d periods. The four treatments were: 1) a control diet (C) with no supplemental B-vitamins; 2) C plus supplemental biotin at 20 mg/d (B); 3) C plus supplemental thiamin (150 mg/d), riboflavin (150 mg/d), pyridoxine (120 mg/d), B12 (0.5 mg/d), niacin (3000 mg/d), pantothenic acid (475 mg/d), folic acid (100 mg/d), and biotin (20 mg/d) (BBVIT1X); 4) C plus supplemental thiamin (300 mg/d), riboflavin (300 mg/d), pyridoxine (240 mg/d), B12 (1.0 mg/d), niacin (6000 mg/d), pantothenic acid (950 mg/d), folic acid (200 mg/d), and biotin (40 mg/d) (BBVIT2X). Intake of DM was increased 0.7 kg/d for B vs. C and BBVIT1X and 1.3 kg/d for B vs. BBVIT2X. Milk yield was increased 1.7 kg/d for B vs. C. For BBVIT1X, milk yield was similar to B and BBVIT2X and tended to be higher than C. Yields of milk protein and lactose but not fat were higher for B than C. For BBVIT1X, milk component yields were similar to B and tended to be higher than C, with the exception of lactose yield where BBVIT1X was higher than C. The objective of Trial 2 was to evaluate DMI and milk yield, composition and component yields by dairy cows fed diets supplemented with either 40 mg/d biotin or the B-vitamin blend (BBVIT1X) compared to cows supplemented with 20 mg/d dietary biotin. Neither the 40 mg/d biotin treatment nor the B-vitamin blend enhanced lactation performance over the 20 mg/d biotin treatment. Biotin efficacy in short-term trials suggests that biotin may improve milk yield directly via effects on intake and (or) nutrient metabolism rather than indirectly via improved hoof health. More research is needed to determine the mode of action for supplemental dietary biotin.