Abstract Context The hypothalamus-pituitary-adrenal (HPA) axis displays a diurnal rhythm. However, little is known about its development in early life. Objective To describe HPA-axis activity and study possible influencing factors in 1-month-old infants. Design Observational. Setting Amsterdam University Medical Center, location VU University Medical Center (VUMC), and Onze Lieve Vrouwe Gasthuis (OLVG), Amsterdam. Participants Fifty-five mother-infant pairs. Interventions Collection of breast milk and infants’ saliva 1 month postpartum for analysis of glucocorticoids (GCs; ie, cortisol and cortisone) using liquid chromatography– tandem mass spectrometry. Main Outcome Measure GC rhythm in infants’ saliva and associations with vulnerability for maternal psychological distress (increased Hospital Anxiety and Depression Scale [HADS] score) or consultation at the Psychiatric Obstetric Pediatric (POP clinic), season at sampling, sex, and breast milk GC rhythmicity analyzed with SigmaPlot 14.0 software (Systat Software, San Jose, CA, USA) and regression analyses. Results A significant biphasic GC rhythm was detected in infants, with mean peaks [standard error of the mean, SEM] at 6:53 am [1:01] and 18:36 pm [1:49] for cortisol, and at 8:50 am [1:11] and 19:57 pm [1:13] for cortisone. HADS score, POP consultation, season at sampling, and sex were not associated with the infants’ GC rhythm. Breast milk cortisol maximum was positively associated with infants’ cortisol area-under-the-curve (AUC) increase and maximum. Higher breast milk cortisone AUC increase, AUC ground, and maximum were associated with an earlier maximum in infants. Breast milk and infant GC concentrations were associated between 6:00 am and 9:00 am. Conclusions A biphasic GC rhythm, peaking in the morning and evening, was seen in 1-month-old infants at a group level. Breast milk GC parameters might be associated with the infants’ GC rhythm, possibly caused by a signaling effect of breast milk GCs, or as an associative effect of increased mother-infant synchrony. These results contribute to an increased understanding of early life HPA-axis development.