Objective: Cesarean section rates are increasing in developed countries and could be performed as an emergency or elective procedure. Our research aim was to determine whether elective cesarean section influences the melatonin content, the main circadian hormone, in human milk. Methods: Twenty-one women after vaginal delivery and 18 women after elective cesarean section were included. Only healthy mothers with normal newborns exclusively breastfed were recruited. Two samples of human milk were collected for each woman at three stages of lactation: colostrum, transitional milk, and mature milk; at each stage, one daytime sample and another nighttime sample were obtained. In total, 228 milk samples were studied. The melatonin content was analyzed by enzyme-linked immunosorbent assay. Results: Melatonin rhythmicity with higher melatonin content at night was maintained at each of the three stages of lactation, regardless of the type of delivery. A higher melatonin content was found in daytime colostrum after cesarean section with respect to colostrum obtained from mothers after vaginal delivery (30.3 pg/mL versus 14.7 pg/mL, p = 0.020). Melatonin content decreased progressively throughout the course of lactation in both groups. This decrease was significant when comparing transitional milk to colostrum in the cesarean group, both in the daytime (p = 0.016) and nighttime samples (p = 0.048). Conclusions: Cesarean section is associated with an increase in daytime colostrum melatonin. No difference was observed in mature milk with respect to vaginal delivery. Melatonin values in human milk decrease during the first month of lactation and circadian rhythmicity was observed irrespective of the mode of delivery.