Amino sugars represent a major constituent of microbial cell walls (e.g. chitin, peptidoglycan) and they are present in large quantities in soil organic matter (SOM). The factors regulating their turnover in soil, however, are poorly understood. Here we investigated the turnover of glucosamine (GlcN) in comparison to glucose (Glc) and N-acetylglucosamine (GlcNAc) in two agricultural grassland soils. Over the range 0–1 mM, GlcN uptake occurred via a saturable high affinity transport systems reflecting its low solution concentrations and low rates of supply. In contrast, Glc uptake was characterised by a non-saturable much lower affinity transport system. Of the GlcN-derived carbon (C) taken into the biomass, ca. 90% was used for the production of new cell biomass rather than in respiration. Whilst temperature affected the uptake (Q10 = 1.95) and mineralization (Q10 = 2.32) of GlcN, it did not affect its C use efficiency within the microbial community. We calculated that the average annual flux of GlcN through the soil was 0.01–0.08 g C kg−1 y−1 which equated to 0.1–1.6% of total heterotrophic soil respiration. Microbial use of GlcN was significantly repressed in the presence of sugars (e.g. Glc, sucrose) and N-acetylglucosamine (GlcNAc). We ascribe this to competition at the transport level and due to internal catabolic repression of metabolic pathways involving GlcN within the microbial biomass. Maize (Zea mays L.) roots showed no capacity to take up exogenously applied GlcN at low external concentrations (10 μM) whilst GlcN was rhizotoxic at higher concentrations (EC50 = 49 μM). This suggests that GlcN does not represent a significant source of dissolved organic nitrogen (DON) for plants. The presence of plants did indirectly, however, suppress the use of GlcN by the rhizosphere microbial community. Our work highlights the importance of GlcN in soil C and N cycling, however, we also raise concerns over its importance relative to that of GlcNAc which our evidence suggests plays a more prominent role in soil C and N cycling.