Purpose: Soil acidification influences competitive N uptake between plants and microorganisms. The mechanisms by which soil acidification affects competition between maize and microorganisms for organic N must be determined to understand N cycling and adjust the forms and levels of N fertilisation. Methods: The uptake of glycine, mineral N after glycine decomposition, and NH4+ by maize and microorganisms was investigated using 13C and 15N labelling. Microbial community composition biomarkers were analysed using phospholipid fatty acid (PLFA) analysis. Mineralisation of organic N was monitored via CO2 production, and gross NH4+/NO3− production and consumption was assessed using 15N pool dilution. Results: Soil acidification (pH from 7.6 to 5.1) increased the intact glycine uptake by maize roots (from 0.7 to 2.4% of added 15N) but decreased its uptake by microorganisms (from 32 to 2.4% of added 15N). Soil acidification altered the microbial community composition: the PLFA of arbuscular mycorrhizal fungi and anaerobes decreased by 6- and 1.5-fold, respectively. Soil acidification reduced the decomposition rates of proteins, peptides, and amino acids as indicated by the CO2 release. This corresponded to a gross NH4+ production increase by 1.3-fold and a gross NO3− production decrease by 97%, compared with soil at pH 7.1. Conclusions: Acidification led to decreased microbial biomass, shift in the microbial community, and the strong decrease (10–15-fold) in amino acid uptake by microorganisms, and was beneficial to maize plants, which assimilated 2.4% of the N added as glycine. However, these quantities of N are insufficient for a substantial increase in the N nutrition of the plants. © 2022, The Author(s), under exclusive licence to Springer Nature Switzerland AG.