A quarter of all anthropogenic methane emissions in the United States are from enteric fermentation, primarily from ruminant livestock. This study was undertaken to test the effect of a methane inhibitor, 3-nitrooxypropanol (3NOP), on enteric methane emission in lactating Holstein cows. An experiment was conducted using 48 cows in a randomized block design with a 2-wk covariate period and a 12-wk data collection period. Feed intake, milk production, and fiber digestibility were not affected by the inhibitor. Milk protein and lactose yields were increased by 3NOP. Rumen methane emission was linearly decreased by 3NOP, averaging about 30% lower than the control. Methane emission per unit of feed dry matter intake or per unit of energy-corrected milk were also about 30% less for the 3NOP-treated cows. On average, the body weight gain of 3NOP-treated cows was 80% greater than control cows during the 12-wk experiment. The experiment demonstrated that the methane inhibitor 3NOP, applied at 40 to 80 mg/kg feed dry matter, decreased methane emissions from high-producing dairy cows by 30% and increased body weight gain without negatively affecting feed intake or milk production and composition. The inhibitory effect persisted over 12 wk of treatment, thus offering an effective methane mitigation practice for the livestock industries.