The emission of enteric methane from dairy cows constitutes a large part of the contribution from agriculture to global warming. The new feed additive “Compound X”, at present under patenting, has been shown to reduce methane emission derived from rumen fermentation in in vitro studies and in one in vivo pilot study with dairy cows. This study aimed to investigate the dose-response effects of “Compound X” on enteric methane emission, milk yield, and feed intake of dairy cows. The experiment was conducted as a 4 × 4 Latin square design using four lactating rumen, duodenal, and ileal cannulated Danish Holstein dairy cows. Four different levels (CON, LOW, MEDIUM, and HIGH) of “Compound X” were supplemented intra-ruminally twice daily to the cows over a period of 14 days. Gas exchange was measured the last 4 d in each period, using four individual transparent respiration chambers based on open-circuit indirect calorimetry. Additionally, milk yield and dry matter intake (DMI) were recorded. Data were analyzed using the mixed procedure of R. Methane emission (L/d) was significantly lower for MEDIUM and HIGH compared to CON. MEDIUM decreased emission by 47%, while HIGH reduced emission by 79%. However, DMI and milk yield were also significantly lower for HIGH compared with CON. The two highest dosages, MEDIUM and HIGH, significantly lowered methane yield (L/kg DMI) by 35% and 79%, respectively. Methane intensity (L/kg ECM) was also significantly lower for MEDIUM and HIGH compared to the two remaining levels. MEDIUM reduced the intensity by 48% and HIGH reduced the intensity by 73% compared to CON. Thus, it can be concluded that intra-ruminal supplementation with MEDIUM or HIGH levels of “Compound X” had a large reducing effect on methane emission. However, HIGH also significantly lowered DMI and milk yield.