Nitric oxide (NO) is essential for plant growth and development, as well as interactions with abiotic and biotic environments. Its importance for multiple functions in plants means that tight regulation of NO concentrations is required. This is of particular significance in roots, where NO signalling is involved in processes, such as root growth, lateral root formation, nutrient acquisition, heavy metal homeostasis, symbiotic nitrogen fixation and root-mycorrhizal fungi interactions. The NO signal can also be produced in high levels by microbial processes in the rhizosphere, further impacting root processes. To explore these interesting interactions, in the present review, we firstly summarize current knowledge of physiological processes of NO production and consumption in roots and, thereafter, of processes involved in NO homeostasis in root cells with particular emphasis on root growth, development, nutrient acquisition, environmental stresses and organismic interactions.