Brazilian coffee cultivation has been expanding into areas of low fertility soils, many times subjected to low water availability, where the use of nitrogen fertilizers is high. To evaluate the effect of water stress on nitrate uptake and on the expression of genes that encode the nitrate uptake and assimilation we have done three experiments with Coffea arabica L. plants. The first was an 8 × 2 factorial (eight coffee cultivars x with or without water stress), with three replicates. We obtained the kinetic parameters of nitrate uptake. The second and third experiments consisted of 2 × 2 (two cultivars x with or without water stress) factorials. One of them was conducted with adequate supply of nitrogen and the other with omission of N. The transcriptional profiles of the genes NRT1.2, NRT3.2, NIA2, GLT, and GLN1.3 were analyzed. Water stress decreased Vmax (maximum velocity of absorption), Km (Michaelis-Menten constant), and Cmin (external concentration at which net uptake of ions is zero) in all cultivars. The cultivar Catuaí Amarelo IAC 62 stood out with high Vmax and low Km in both conditions, and the Cultivar Mundo Novo IAC379-19, although with low Vmax, had low Km and low Cmin, without changes under water stress. In plants under water stress, the genes NRT1.2, NRT3.2, NIA2, and GLT showed higher differential transcription in root systems. GLN1.3 gene had higher relative expression in the shoots. Under water stress, the relative expression of these genes was higher in Mundo Novo IAC 379-19 than in Catuaí Amarelo IAC 62 in both nitrogen doses.