A study was conducted to examine the effects of supplemental dietary protease enzymes on production variables in dairy cattle. Ninety-six multiparous lactating Holstein cows (624 +/- 62 kg body weight and 154 +/- 104 days in milk) were blocked according to parity, days in milk, and previous milk production and randomly assigned to a control total mix ration (TMR) or a TMR containing a blend of supplemental protease enzymes (PE; 4 g/cow/d) in a crossover design with two 21-day experimental periods. Daily pen milk yield and dry matter intake (DMI) were recorded and milk composition from all cows was determined on d 15, 17. 19 and 21 of each period. There was no treatment effect on milk yield (37.6 kg/d), but supplemental PE-fed cows consumed less DMI (P<0.05) compared to controls and therefore tended to have improved feed efficiency (P=0.06). Feeding supplemental PE decreased blood urea nitrogen (P<0.05) compared to the control cows. However, feeding PE had no effect on milk fat and protein content but tended (P=0.08) to increase milk lactose concentration and tended (P=0.10) to decrease milk urea nitrogen levels and somatic cell score. Results indicate that supplemental PE may enhance production efficiency and improve parameters of nitrogen status.