Abstract Context The male hypothalamic-pituitary-gonadal (HPG) axis is transiently active during the first months of life with surging serum concentrations of reproductive hormones. This period, termed minipuberty, appears to be essential for priming testicular function. Despite the central role for male reproductive function, longitudinal data on HPG axis activation in infancy is sparse. Objective To explore the dynamics of HPG hormone activity in healthy male infants, to assess the association of HPG axis activity and testicular volume, and to establish reference curves for serum levels of reproductive hormones. Design Prospective, longitudinal birth cohort (the COPENHAGEN Minipuberty Study, 2016-2018, 1-year follow-up). Setting Population-based. Patients or Other Participants Healthy, male, term, singleton newborns were followed from birth on with repeated clinical examinations including blood sampling during a 1-year follow-up. A total of 128 boys contributed to this study, while 119 participated in the postnatal follow-up. Main Outcome Measures Serum reproductive hormone concentrations and testicular volume. Results Reproductive hormone concentrations showed marked dynamics during the first 6 months of age. Gonadotropins, total testosterone, and insulin-like factor 3 peaked at around 1 month of age. Inhibin B, anti-Müllerian hormone, and testicular volume peaked at around 4 to 5 months. Correlations largely recapitulated typical HPG axis pathways but also differed significantly from adult men. Conclusions We demonstrate a temporal dissociation of Leydig and Sertoli cell activity during male minipuberty and provide reference curves for reproductive hormones.