The protein nutrition of dairy cows is of great importance because of its direct influence on milk production, reproductive efficiency, and feeding cost. Eight first-lactation Holstein cows were randomly assigned to two contemporary 4 × 4 Latin squares in a 2 × 2 factorial design to evaluate the effects of replacing soybean meal with yeast-derived microbial protein (YMP) as a protein source (0% or 1.5% of dry matter (DM)) and its combination with slow-release urea (SRU; 0% or 0.75% of DM) on DM intake and milk production and composition, as well as blood parameters and nitrogen balance. Each experimental period lasted 28 days, with 21 days of adaptation and 7 days of data collection. The diets were formulated to attend the nutritional recommendations of the National Research Council and consisted of 49% forage (47% corn silage and 2% Tifton hay) and 51% concentrate, with 16.8% CP and 1.6 Mcal net energy for lactation/kg DM. For diets without YMP, the inclusion of SRU decreased DM intake, milk production as well as N intake and balance, but did not affect efficiency of production, milk composition or most of blood parameters. On the contrary, for diets with YMP, DM intake and milk production were increased by inclusion of SRU, while minor effects were observed for milk efficiency and composition, blood parameters as well as N intake, excretion and balance. When diets with SRU were compared, the inclusion of YMP increased DM intake, 4% fat-corrected milk, and N intake and balance (P<0.05), with no differences in milk production (kg/day), milk energy, efficiency of milk production or most of the blood parameters. For diets without SRU, YMP inclusion decreased DM intake, milk production, milk energy, N intake, fecal N and N balance (P<0.05), with no effects on milk efficiency and composition, or most of blood parameters. In conclusion, the use of YMP, SRU or both as partial substitutes of soybean meal in the diet of lactating cows has no negative effects on productivity parameters.