Nitrogen is a very important nutrient in grass maintenance fertilization and therefore must be applied at the appropriate moment. The objective of this study was to identify the most responsive moment to nitrogen fertilization and to verify if root mass and the content of carbohydrates and nitrogen in roots influence the moment of fertilization in cultivars of Megathyrsus maximus (syn. Panicum maximum). This study was carried out simultaneously in a greenhouse using a completely randomized design, with sixteen treatments and five replications, in a 4×4 factorial design. The treatments consisted of four intervals between cultivar defoliation and nitrogen fertilization (0, 3, 6 and 9 days) and four Megathyrsus maximus cultivars, Mombasa, BRS Zuri, BRS Quenia and BRS Tamani, which were evaluated in five regrowth cycles. No difference in forage mass was observed among cultivars when fertilization was performed on days zero, three and nine after harvesting. On day nine, Mombasa showed a higher forage mass compared to BRS Tamani. Nitrogen content in the roots of Zuri decreased when fertilization was performed on the third day after defoliation, remaining constant in the other fertilization intervals. A linear reduction in root starch in BRS Zuri was observed, while in Mombasa cultivars, a linear increase was observed when fertilization was performed nine days after harvesting. Thus, nitrogen fertilization of BRS Tamani should be carried out closer to defoliation, while Mombasa, BRS Zuri and BRS Quenia can be fertilized up to nine days after harvesting, which results in greater flexibility regarding the moment of nitrogen fertilization.