Nitrogen (N) is often applied to soil surface during the grapevine cycle, increases N losses. One way to reduce N losses lies on its application method, however how N dose and application mode impact on grapevines remain poorly investigated. The aim of this study is to evaluate the root growth, as well as grape must yield and composition, in a sandy soil and subjected to different N application doses and modes under subtropical climate conditions. Grapevines were implanted in 2011 and, since 2014, they were subjected to surface (N-Surf), and fertigation (N-Fert) N application, at rates of 0, 40 and 100 kg N ha−1 year−1. Leaf N concentration, yield, must quality parameters and root system morphology were evaluated in the 2016/2017 and 2017/2018 seasons. The application of N in both rates (40 and 100 kg N ha−1 year−1), in the N-Surf and N-Fert methods, increased the surface area of root, number of living roots and root production. The application of N rates, in both N application modes, provided adequate N supply to the soil and increased root system development, which contributed to the increase of soil N uptake by plants, higher N concentration in leaves and greater grape yield. However, N supply decreased the quality of the must, as observed in decreased TSS and total anthocyanins values and in increased total titratable acidity values, which are not desirable for red wine making processes.