The rumen simulating technique (RUSITEC) was used to evaluate the effect of dietary inclusion of amaranth seeds on the rumen fermentation and the concentration of fatty acids (FA) in fermentation fluid. Four fermentation vessels of the RUSITEC were used. The control diet (C) consisted of 70% meadow hay and 30% barley meal, whereas 10% of barley meal was replaced by milled (Amill), mechanically ground (Agr), and ground after heating in a microwave amaranth seeds (Aheat+gr) in other experimental vessels, respectively. All diets were isonitrogenous (11% of crude protein). With degradability of dry matter (DM) and acid-detergent fibre (ADF) not affected (P > 0.05), degradability of neutral detergent fibre (NDF) was significantly decreased by the inclusion of Amill (P < 0.001) and Aheat+gr (P < 0.05). Heating, compared to milling, decreased degradation of crude protein (CP) and addition of amaranth seeds generally increased production of microbial nitrogen. Production of fermentation gasses, methane, and total volatile fatty acids (VFA) was not affected by changes in composition of diets. Addition of amaranth seeds in a milled form (Amill) caused a significant decrease in concentrations of acetate and propionate and growth in concentrations of butyrate, iso-valerate, and caproate. No effect (P > 0.05), compared to C, was found on the percentage of saturated or unsaturated FA, but changes in concentrations of some FA were observed. An absence of any detrimental effects on ruminal fermentation patterns indicated that grain amaranth seeds can partially substitute for the barley in ruminant nutrition.