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Incorporation of nitrogen (N) into soil microbial protein is central to the soil N cycle to mitigate N losses and support plant N supply. However, the effect of factors, such as water filled pore space (WFPS), which influence inorganic N transformations and losses, and thus microbial incorporation, are only poorly understood. This work aimed to bridge this gap, using compound-specific 15N-stable isotope probing to quantify microbial assimilation into the largest defined soil organic N pool, protein-N. This approach applied differentially 15N-labelled ammonium nitrate (NH 4 NO 3) to an unfertilised UK grassland in a soil mesocosm study over 10 days. The soil microbial community showed a strong preference for NH 4 + over NO 3 −, which varied with WFPS (85% > 55% > 70%). This preference decreased for amino acids further in biosynthetic proximity to the transamination step in amino acid biosynthesis. Combined incorporation of NH 4 + and NO 3 − increased total hydrolysable amino acid-N concentration linked to WFPS (55% ∼ 85% > 70%). Incorporation rates of applied 15N showed the same trend as NH 4 + preference with WFPS (85% > 55% > 70%), which is related to microbial activity and nutrient mobility. Despite differences in incorporation, when normalised to soil available N, incorporation was comparable in the short-term. Mechanistic control of WFPS via assimilation into the largest soil organic N pool is important to mitigate potential positive feedbacks to N losses and support N supply to plants. • Total hydrolysable amino acid concentration increased at 55% and 85% WFPS. • Incorporation into the soil microbial protein pool showed a preference for NH 4 + over NO 3 −. • Preference of amino acids depended on biosynthetic proximity to transamination. • 15N incorporation into soil microbial protein was influenced by WFPS (85% > 55%>70%). [ABSTRACT FROM AUTHOR]