Biotin supplementation resulted in an increased milk production in multiparous cows during weeks 2 to 6, but the effect was no more significant between 7 and 17 weeks of lactation. Milk protein percent was decreased by 0.1% in multiparous cows. Milk fat content was not affected by biotin, and milk fat daily production tended to increase during early lactation. In milk fat, biotin supplementation tended to decrease the proportion of fatty acids with less than 16 carbons at week 3, but the daily amount was not affected. Biotin tended to decrease biohydrogenation intermediates, increased C16:1 at week 3, and tended to increase cis-9 C18:1 at weeks 3 and 10. After 7 weeks of lactation, biotin tended to increase blood beta-hydroxybutyrate in multiparous cows with values remaining in a normal range, and decreased plasma glucose in primiparous cows. These modifications of plasma parameters, milk protein content and profile of milk fatty acids could be due to a higher lipid mobilisation from adipose tissue driven by the increased milk production.