Exogenous fibrolytic enzymes have been shown to be a promising way to improve feed conversion efficiency (FCE). β-Mannanase is an important enzyme digesting the polysaccharide β-mannan in hemicellulose. Supplementation of diets with β-mannanase to improve FCE has been more extensively studied in nonruminants than in ruminants. The objective of this study was to investigate the effects of β-mannanase supplementation on nutrient digestibility, FCE, and nitrogen utilization in lactating Holstein dairy cows. Twelve post-peak-lactation multiparous Holstein cows producing 45.5±6.6kg/d of milk at 116±19.0d in milk were randomly allotted to 1 of 3 treatments in a 3×3 Latin square design with 3 periods of 18d (15d for adaptation plus 3d for sample collection). All cows were fed the same basal diet and the 3 treatments differed only by the β-mannanase dose: 0% dry matter (DM; control), 0.1% of DM (low supplement, LS), and 0.2% of DM (high supplement, HS) supplemented to the basal diet. Supplementation of β-mannanase enzyme at the LS dose reduced dry matter intake (DMI) but did not affect milk yield or milk composition. Cows receiving LS produced 90g more milk per kg of DMI compared with control cows. Somatic cell count (SCC) in milk was lower for cows fed the LS diet compared with cows fed control diets. Cows fed LS diet had lower DM, organic matter and crude protein digestibility compared with cows fed control diets. Starch, neutral detergent fiber, and acid detergent fiber digestibility were not affected by LS. Milk yield, DMI, SCC, and nutrient digestibility did not change for HS. Despite the reduced crude protein digestibility, reduced N intake led to similar fecal N excretions in LS cows and control cows (234 vs. 235g/cow per day). Urinary N excretions remained similar between enzyme-fed and control cows (~190g/cow per day), although the percentage of N intake partitioned to urinary N tended to be greater in LS than in control cows (31 vs. 27%). Cows fed LS significantly improved the percentage of apparently absorbed N partitioned to milk protein N (42 vs. 38%). When supplemented at 0.1% of dietary DM, β-mannanase can improve FCE and lower the SCC of dairy cows without affecting milk yield, milk composition, or total manure N excretions of dairy cows.