Nitrogen use efficiency is an important index in ruminants and can be indirectly evaluated through the N isotopic discrimination between the animal and its diet ( Δ 15 N animal-diet ). The concentration and source of N may determine both the extent of the N isotopic discrimination in bacteria and N use efficiency. We hypothesised that the uptake and release of ammonia by rumen bacteria will affect the natural 15 N enrichment of the bacterial biomass over their substrates ( Δ 15 N bacteria-substrate ) and thereby further impacting Δ 15 N animal-diet . To test this hypothesis, two independent in vitro experiments were conducted using two contrasting N sources (organic vs inorganic) at different levels either in pure rumen bacteria culture incubations (Experiment #1) or in mixed rumen cultures (Experiment #2). In Experiment #1, tryptone casein or ammonium chloride were tested at low (1 mM N) and high (11.5 mM N) concentrations on three rumen bacterial strains ( Fibrobacter succinogenes , Eubacterium limosum and Xylanibacter ruminicola ) incubated in triplicate in anaerobic batch monocultures during 48h. In Experiment #2 mixed rumen cultures were incubated during 120 h with peptone or ammonium chloride at five different levels of N (1.5, 3, 4.5, 6 and 12-mM). In experiment #1, Δ 15 N bacteria-substrate was lowest when the ammonia-consumer bacterium Fibrobacter succinogenes was grown on ammonium chloride, and highest when the proteolytic bacterial strain Xylanibacter ruminicola was grown on tryptone. In experiment #2, Δ 15 N bacteria-substrate was lower with inorganic (ammonium chloride) vs organic (peptone) N source. A strong negative correlation between Δ 15 N bacteria-substrate and Rikenellaceae_RC9_gut_group , a potential fibrolytic rumen bacterium, was detected. Together, our results showed that Δ 15 N bacteria-substrate may change according to the balance between synthesis of microbial protein from ammonia versus non-ammonia N sources and confirm the key role of rumen bacteria as modulators of Δ 15 N animal-diet .