Abstract The effect of linseed oil (LSO) supplementation on total-tract and ruminal nutrient digestibility, N metabolism, and ruminal fluid characteristics was investigated in dairy cows fed diets containing different forage to concentrate ratios (F:C). The experimental design was a 4×4 Latin square with 2×2 factorial arrangement of treatments. Four lactating Holstein cows were fed a forage-rich diet without LSO (F; F:C = 65:35, dry matter basis), a forage-rich diet with LSO (FO; F:C = 65:32, 3% LSO), a concentrate-rich diet without LSO (C; F:C = 35:65), or a concentrate-rich diet with LSO (CO; F:C = 35:62, 3% LSO). Total-tract digestibility of DM and OM was greater with supplemental LSO. A tendency for greater total-tract digestibility of NDF and ADF also was observed in cows fed LSO. Ruminal digestibility of NDF or ADF decreased when CO was fed compared with C. In contrast, feeding FO increased NDF or ADF digestibility compared with F. Although ruminal starch digestion was nearly complete with all diets, digestibility was greater when cows were fed C or CO compared with F or FO. Bacterial N flow to the duodenum decreased when FO was fed compared with F. In contrast, feeding CO increased bacterial-N flow compared with C. Neither F:C nor LSO supplementation affected ruminal pH or total VFA concentration in ruminal fluid. However, molar proportion of propionate was greater with C or CO compared with F or FO and increased with LSO supplementation regardless of F:C. Molar proportion of n-butyrate decreased with LSO supplementation. Total protozoal numbers in ruminal fluid decreased markedly only when CO was fed. Overall, data show that feeding LSO had no negative effects on total-tract digestion in dairy cows but may decrease ruminal fiber digestibility when fed with high-concentrate diets. The widely spread idea that LSO decreases digestibility, arising from studies with sheep, did not seem to apply to lactating cows fed 3% LSO.