An experiment of 3 x 3 Latin square design was conducted to evaluate the effect of lupin seeds (Lupinus albus, var. Amiga) as a substitute for soybean meal in a diet of lactating dairy cows. For the experiment, 30 high yielding dairy cows were divided into three well balanced groups, 10 cows in each group. Three types of diets which differed in the content of soybean meal were prepared. The control diet contained 100% of soybean meal, whereas there was replaced about 30% and 50% of soybean meal by lupin seeds in the diets P30 and P50, respectively. Each period of the experiment lasted four weeks with three preparatory weeks and at the fourth week of sampling of milk, rumen fluid and blood (from caudal vein). There were no statistically conclusive differences (P > 0.05) in dry matter intake, milk production and milk composition. Lupin seeds can effectively replace as much as 50% of soybean meal in a diet of dairy cows with maintenance the same level of milk production.