The effects of nitrogen fertilization on protein content, N uptake and N use efficiency of grain for six spring wheat cultivars were evaluated over a N application range of 0–200 kg ha−1, under two moisture supply levels, on Black Chernozemic soils in Manitoba. Moisture supply influenced protein content, protein yield, and grain N use efficiency (NUE) of applied fertilizer. Increased moisture supply lowered protein content and increased protein yield and NUE. Increasing N level increased protein, N uptake and decreased NUE, but effects depended on moisture supply. Cultivar differences occurred, especially at the higher moisture level.Key words: Protein, Triticum aestivum L., nitrogen uptake, nitrogen use efficiency, moisture