The aim of this study was to assess biomass production of pearl millet as a function of levels and phases of defoliation. The experiment was carried out in a protected environment at the Fazenda União experimental farm in the municipality of Nova Xavantina, MT, Brazil, in 8 dm 3 capacity pots in soil classified as dystrophic red Latosol. The experimental design used was randomized blocks in a 5 × 3 + 1 factorial arrangement, with five phases of defoliation (three expanded leaves, six expanded leaves, flag leaf, booting, full flowering), three levels of defoliation (33, 66 and 99%), and an additional treatment without defoliation, with three replications. The pearl millet cultivar ADR 300 was used. Plant height, number of tillers, total dry biomass of the seed heads, and total dry biomass of the plants during the leaf rolling phase was assessed. It was observed that plant height at all levels and phases of defoliation was greater than the control. For number of tillers, a reduction was observed in the booting phase and full flowering phase at all levels of defoliation; and three expanded leaves and flag leaf phases for the levels of 66 and 99% defoliation. Total biomass of the panicle at all levels and phases of defoliation was less than the control. For total dry biomass, there was a statistical difference in relation to the control when the plants were subjected to 33% defoliation in the booting phase and full flowering phase, and 66 and 99% defoliation in all the phases. A fall in production of total dry biomass of the plant greater than 53% was observed with total elimination of the leaves during the flag leaf phase.