AbstractWe evaluated the effects of incremental amounts of ground flaxseed (GFX) on diversity and relative abundance of ruminal microbiota taxa, enteric methane (CH4) emissions, and urinary excretion of purine derivatives (PD) in lactating dairy cows in a replicated 4 × 4 Latin square design. Twenty mid-lactation Jersey cows were used in the study. Of these 20 cows, 12 were used for ruminal sampling, 16 for enteric CH4measurements, and all for spot urine collection. Each period lasted 21 d with 14 d for diet adaptation and 7 d for data and sample collection. Diets were formulated by replacing corn meal and soybean meal with 0%, 5%, 10%, and 15% of GFX in the diet’s dry matter. Ruminal fluid samples obtained via stomach tubing were used for DNA extraction. Enteric CH4production was measured using the sulfur hexafluoride tracer technique. Diets had no effect on ruminal microbiota diversity. Similarly, the relative abundance of ruminal archaea genera was not affected by diets. In contrast, GFX decreased or increased linearly the relative abundance of Firmicutes (P< 0.01) and Bacteroidetes (P< 0.01), respectively. The relative abundance of the ruminal bacteriaRuminococcus(P< 0.01) andClostridium(P< 0.01) decreased linearly, and that ofPrevotella(P< 0.01) andPseudobutyrivibrio(P< 0.01) increased linearly with feeding GFX. A tendency for a linear reduction (P= 0.055) in enteric CH4production (from 304 to 256 g/d) was observed in cows fed increasing amounts of GFX. However, neither CH4yield nor CH4intensity was affected by treatments. Diets had no effect on the urinary excretion of uric acid, allantoin, and total PD. Overall, feeding GFX decreased linearly the relative abundance of the ruminal bacterial generaRuminococcusandClostridiumand enteric CH4production, but no change was seen for CH4yield and CH4intensity, or urinary excretion of total PD, suggesting no detrimental effect of GFX on microbial protein synthesis in the rumen.