The polyamine content of human breast milk, which is the first exogenous source of polyamines for the newborn, can be affected by several factors associated with the mother, the infant, or breastfeeding itself. The aim of this study was to evaluate the influence of different breastfeeding factors on the polyamines found in human milk. For this study, a cohort of 83 mothers was considered for up to 4 months, and a subgroup of 33 mothers were followed during the first six months of breastfeeding. Two breast milk samples were collected at each sampling point (foremilk and hindmilk) and the polyamine content was determined by UHPLC-FL. Polyamine levels varied considerably between the mothers and tended to decrease over time. Putrescine was the minor polyamine, whereas spermidine and spermine contents were very similar. The concentrations of the three polyamines were significantly higher in hindmilk than foremilk ( < 0.001). Spermidine and spermine levels decreased significantly through the lactation progress ( < 0.05). Finally, slightly higher levels of polyamines were observed in the milk of mothers providing partial, rather than full, breastfeeding, although the differences were not significant. The polyamine content in human milk was found to change during a single feed (foremilk versus hindmilk) and as lactation progressed, mainly in response to the specific circumstances of the newborn.