Abstract <p>Molybdenum (Mo) is an important microelement for animals and humans. The requirements of productive animals for this element are not well defined, in particular for beef cattle, since its deficiency has not been recorded under practical feeding conditions. There is limited evidence that inclusion of molybdenum in the diet of sheep and cattle can improve diet digestibility. The aim of the study was to investigate the effect of Mo on the rumen microbiota and the course of metabolic processes in the rumen using an in vitro method. In the experiment, Mo nanoparticles (NPs) (size 50 nm, 99.7% molybdenum, 0.3% O₂, 14 m²/g <i>S</i>_sp, obtained by plasma-chemical synthesis) were used at doses of 1.0, 1.7, and 2.0 mg/kg dry matter (DM). Mill offal was used as a feed substrate (in the control without the inclusion of NPs). The study of metabolic processes in the rumen of ruminants was carried out by the in vitro method using the ANKOM DaisyII incubator (modifications D200 and D200I) according to a specialized technique. The use of supraphysiological doses of molybdenum in the artificial rumen system can disrupt the balance of microorganisms, reducing the number of ciliates by 9.3–15.4% and the biomass of protozoa in the rumen contents by 18.2–22.7%, while stimulating the growth of bacteria by 19.2–30.8% in doses not exceeding 1.7 mg/kg DM. The inclusion of various doses of molybdenum contributed to an increase in the digestibility of the feed substrate DM by up to 5%, the level of VFA by 12.4–21.1%, and total nitrogen in the rumen fluid by 21.1–1.1% relative to the control values, which allows us to consider this element as a component of a premix for ruminants in order to stimulate metabolism and increase productivity.</p>