Background The human milk (HM) composition reflects the nutritional status and dietary intake of the nursing mother, especially regarding the fatty acids (FA) intakes. Evidence has shown that Brazil nut is a good source of FA (mono and polyunsaturated) and selenium. Thus, this study tests the hypothesis that the consumption of this nut during lactation can modify the nutritional profile of HM, increase its energy density and selenium content, and improve the lipid profile. Therefore, this study aimed to describe the design and methods of the Randomized Clinical Trial (RCT): Effect of maternal dietary supplementation with Brazil nut (Bertholletia excelsa) on the nutritional profile of HM. Method/Design It is a crossover study, conducted with mother-infant dyads (with infants born at full-term) recruited from a maternity hospital and followed up to the third month postpartum in a pediatric office. Sociodemographic, lifestyle, obstetric, and birth data were collected at 7 days postpartum. Maternal nutrition assessment data (anthropometry, body composition, food intake, biochemical tests), HM samples, and infant anthropometric and health assessment data were collected on average at 37, 52, 75, and 90 days postpartum. The nursing mothers were randomly allocated into two groups: group (1) started with supplementation of 1.5 g/day Brazil nut (case) for 15 days, followed by 15-day washout period; after the washout, the group was then considered as control and received follow-up monitoring for another 15 days; group (2) started as control for 15 days, which was followed by a washout period of 15 days, then they initiated supplementation with Brazil nut for 15 days (case).Discussion To the best of our knowledge, this is the first RCT to evaluate the effect of maternal Brazil nut supplementation on HM nutritional profile. This is a promising research area that can provide findings which will contribute to improving health and nutrition of infants, especially premature infants, admitted to Neonatal Intensive Care Units.