A long-term study was conducted to evaluate the effects of 3-nitrooxypropanol (3-NOP) on methane (CH₄) emissions and milk production in Holstein-Friesian dairy cows over a full year. Sixty-four cows were divided into pairs based on parity, expected calving date, and milk yield. The study included an adaptation period, a covariate period, and a 48-week trial phase. Cows were randomly assigned to two diets: one containing 69.8 mg/kg DM of 3-NOP and another with a placebo. The study found that 3-NOP reduced CH₄ production by 21%, 20%, and 27% for CH₄ production, yield, and intensity, respectively. Additionally, 3-NOP increased energy-corrected milk (ECM) and fat- and protein-corrected milk (FPCM) yields by 6.5%. Milk fat and protein content, as well as feed efficiency, were also improved. The effectiveness of 3-NOP was influenced by the diet composition and lactation stage. The mitigation potential of 3-NOP was greater in diets with lower concentrate levels and higher forage content. The study also found that 3-NOP had a positive effect on milk production characteristics without affecting dry matter intake (DMI) or body weight. However, the efficacy of 3-NOP decreased over time but not continuously. The study highlights the importance of diet composition in determining the effectiveness of 3-NOP, suggesting that changes in diet composition can significantly impact its mitigation potential. Further research is needed to fully understand the long-term effects of 3-NOP on CH₄ emissions and its interaction with varying diet compositions.A long-term study was conducted to evaluate the effects of 3-nitrooxypropanol (3-NOP) on methane (CH₄) emissions and milk production in Holstein-Friesian dairy cows over a full year. Sixty-four cows were divided into pairs based on parity, expected calving date, and milk yield. The study included an adaptation period, a covariate period, and a 48-week trial phase. Cows were randomly assigned to two diets: one containing 69.8 mg/kg DM of 3-NOP and another with a placebo. The study found that 3-NOP reduced CH₄ production by 21%, 20%, and 27% for CH₄ production, yield, and intensity, respectively. Additionally, 3-NOP increased energy-corrected milk (ECM) and fat- and protein-corrected milk (FPCM) yields by 6.5%. Milk fat and protein content, as well as feed efficiency, were also improved. The effectiveness of 3-NOP was influenced by the diet composition and lactation stage. The mitigation potential of 3-NOP was greater in diets with lower concentrate levels and higher forage content. The study also found that 3-NOP had a positive effect on milk production characteristics without affecting dry matter intake (DMI) or body weight. However, the efficacy of 3-NOP decreased over time but not continuously. The study highlights the importance of diet composition in determining the effectiveness of 3-NOP, suggesting that changes in diet composition can significantly impact its mitigation potential. Further research is needed to fully understand the long-term effects of 3-NOP on CH₄ emissions and its interaction with varying diet compositions.