Nitrogen fertilization, in addition to increasing biomass production, can also positively influence the chemical composition of a forage plant. The aim of this research was to evaluate the yield and chemical composition of Megathyrsus maximus cv. Massai grass under five nitrogen fertilization levels (0, 150, 300, 450 and 600 mg N dm-3 soil, equivalent to 0, 300, 600, 900 and 1,200 kg ha-1 year-1, respectively) and during growth cycles (establishment and regrowth cycles) in a greenhouse. A completely randomized design with split-plot arrangement was adopted in which the five nitrogen levels were the plots and the cycles were the subplots. The total forage biomass increased with the nitrogen fertilization levels in the three growth cycles (28.4 g pot-1, 32.0 g pot-1 and 29.8 g pot-1 for establishment, regrowth 1 and regrowth 2, respectively, at the level of 600 mg N dm-3), and it was reduced from establishment to regrowth at lower N levels. The dry matter content was not affected by the N levels. However, this content presented higher values at the regrowth cycle in comparison to the establishment cycle. The crude protein (CP) content increased (7.0% CP at the level of 600 mg N dm-3), while the neutral detergent fiber (NDF), acid detergent fiber (ADF) and hemicellulose (HEM) contents all decreased at higher N levels, with the highest CP content being observed at the establishment, and the opposite occurring for NDF, ADF and HEM. Nitrogen fertilization provides positive responses on yield and chemical composition of massai grass.