Updated project metadata. The time-resolved impact of monensin on the active rumen microbiome in a rumen-simulating technique (Rusitec) was studied with metaproteomic and metabolomic approaches. Upon monensin treatment, decreased catabolism linked to fiber degradation was observed by the reduced abundance of proteins assigned to fibrolytic bacteria and glycoside hydrolases, sugar transporters and carbohydrate metabolism. Reduced amounts of ammonium as well as branched-chain fatty acids pointed towards a decreased proteolytic activity. The family Prevotellaceae exhibited increased resilience in the presence of monensin, with a switch of metabolism from acetate to succinate production. Prevotella species harbor a membrane bound electron transfer complex, which drives the reduction of fumarate to succinate, the substrate for propionate production in the rumen habitat. Besides the increased succinate production, a concomitant depletion of methane concentration was observed upon monensin exposure. Our study demonstrates that Prevotella sp. shifts its metabolism successfully in response to monensin exposure and Prevotellaceae represents the key bacterial family stabilizing the rumen microbiota during exposure to monensin.