Effects of rumen protozoa on ruminal fermentation, methane (CH4) emissions and nitrogen (N) retention were studied in twelve crossbred ewes given an oaten chaff diet. Over 10 days sheep were progressively adapted to a diet containing 7% coconut oil distillate to suppress rumen protozoa and then were defaunated using sodium 1-(2-sulfonatooxyethoxy) dodecane (Empicol). Twelve weeks after defaunation treatment, five sheep were inoculated with rumen fluid collected from cannulated sheep to refaunate them and that the effect of re-establishment of rumen protozoa 0, 7, 14 and 21 days following refaunation on ruminal fermentation and CH4 emissions was examined in Experiment 1. As a following study (Experiment 2), feed intake was restricted to 1.5 x ME requirement for maintenance from day 28 to day 43 when dry matter (DM) digestibility, N retention, fermentation and CH4 emissions were compared between defaunated and refaunated sheep. Sheep were scanned through a computed tomography scanner on day 0 and day 28 to estimate reticulo-rumen (RR) weight and carcass composition. It was concluded that refaunated sheep did not have a higher daily CH4 production (DMP, g CH4/day) than did the defaunated cohort within 21 days after refaunation as measured by Greenfeed Emission Monitoring units. Total volatile fatty acid (VFA) concentration and the proportion of propionate in the rumen VFA gradually increased over 21 days following refaunation (Experiment 1), while a change towards higher butyrate and lower acetate proportions was observed after 28 days (Experiment 2; P<0.05). There was a tendency towards a heavier RR weight (P=0.08) and a higher ratio of RR to liveweight in defaunated sheep 28 days after refaunation (P < 0.001), but carcass composition was not affected by refaunation status. Experiment 2 showed defaunated sheep had a 7% lower DMP than did refaunated sheep with an established rumen fauna (P<0.05). Apparent whole-tract N and DM digestibility and microbial crude protein supply were not different between defaunated and refaunated sheep, while energy losses in CH4 (MJ/day) and CH4 as a proportion of gross energy intake were both approximately 8% lower in defaunated sheep. The reduced CH4 emissions achieved by defaunation occurred without altering total VFA, apparent whole-tract N and DM digestibility or ADG.