Two simulated rumens (Rusitecs) were used to assess the effects of flaxseed (FS) and (or) vitamin E (VE) on rumen fermentation, fatty acid (FA) biohydrogenation, and microbial protein synthesis. Ground FS replaced 0% or 15% of barley grain, along with VE at 0 or 1000 IU d⁻¹ in a 2 × 2 factorial experiment. Flaxseed lowered neutral detergent fiber (P = 0.001) and acid detergent fiber (P = 0.01) and increased (P = 0.001) nitrogen (N) disappearance. Flaxseed also increased (P = 0.01) total volatile FA and decreased (P = 0.001) acetate production. When both FS and VE were included, the acetate:propionate ratio decreased (P = 0.04). Biohydrogenation of FA was not influenced by VE, but total FA and C18:0 in effluent were increased (P = 0.001) and C16:0 decreased (P = 0.001) by FS. With VE, total microbial N (MN) was increased (P = 0.001). In the concentrate, production of MN in feed-particle-bound bacteria was increased (P = 0.001) by VE. Vitamin E did not alter FA biohydrogenation but did promote MN production. The stable and relatively high pH in the Rusitec may have prevented the typical shift from C18:1 trans-11 to C18:1 trans-10 with concentrate diets. Future studies simulating subclinical acidosis in the Rusitec may illuminate ruminal mode(s) of action of VE on FA biohydrogenation.