Main conclusion: Non-optimal ammonium levels significantly alter root architecture, anatomy and root permeabilities for water and nutrient ions. Higher ammonium levels induced strong apoplastic barriers whereas it was opposite for lower levels. Application of nitrogen fertilizer increases crop productivity. However, non-optimal applications can have negative effects on plant growth and development. In this study, we investigated how different levels of ammonium (NH4 +) [low (30 or 100 μM) or optimum (300 μM) or high (1000 or 3000 μM)] affect physio-chemical properties of 1-month-old, hydroponically grown rice roots. Different NH4 + treatments markedly altered the root architecture and anatomy. Plants grown in low NH4 + had the longest roots with a weak deposition of suberised and lignified apoplastic barriers, and it was opposite for plants grown in high NH4 +. The relative expression levels of selected suberin and lignin biosynthesis candidate genes, determined using qRT-PCR, were lowest in the roots from low NH4 +, whereas, they were highest for those grown in high NH4 +. This was reflected by the suberin and lignin contents, and was significantly lower in roots from low NH4 + resulting in greater hydraulic conductivity (Lpr) and solute permeability (Psr) than roots from optimum NH4 +. In contrast, roots grown at high NH4 + had markedly greater suberin and lignin contents, which were reflected by strong barriers. These barriers significantly decreased the Psr of roots but failed to reduce the Lpr below those of roots grown in optimum NH4 +, which can be explained in terms of the physical properties of the molecules used and the size of pores in the apoplast. It is concluded that, in rice, non-optimal NH4 + levels differentially affected root properties including Lpr and Psr to successfully adapt to the changing root environment.