Background: Insulin, insulin-like Growth Factor-1 (IGF-1), and obestatin in human milk originate from the circulation. There is also limited knowledge about the influence of body fat on the levels of these hormones in human milk. Research Aim: To determine (1) the influence of body fat on levels of insulin, IGF-1, and obestatin in human milk and serum/plasma during the postpartum period; (2) the changes in the levels of these hormones in human milk and serum/plasma postpartum; and (3) the presence of IGF-1 mRNA in human milk. Methods: In this prospective, longitudinal, observational cohort study, levels of insulin, IGF-1, and obestatin were measured up to 30 days postpartum in milk and serum/plasma of 58 participants with adequate (≤ 32%) or excess (> 32%) total body fat determined by electrical bioimpedance. Student’s t test and repeated-measures analysis of variance were used to evaluate the differences between groups. Pearson’s test was used to analyze the associations. Results: The milk from participants with excess body fat had higher insulin and IGF-1 levels and lower obestatin levels than that of participants with adequate body fat at 3–7, 14–15, and 30 days postpartum (adjusted p < .001). The levels of insulin, IGF-1, and obestatin were significantly higher in human milk than in serum/plasma ( p < .05) and correlated with maternal body fat ( p < .001). Conclusions: Maternal body fat was associated with elevated insulin and IGF-1 levels and decreased obestatin levels in human milk up to 30 days postpartum.