BACKGROUND In a recent study, we performed a complete structural characterization of glycosaminoglycans (GAGs) in human mature milk. However, no data are available on the total content of GAGs in human milk from healthy mothers having delivered term or preterm newborns. OBJECTIVE In this study, we evaluated the total content of GAGs in pooled milk from healthy mothers having delivered term or preterm newborns during the first month of lactation. METHODS Highly specific and sensitive analytical approaches were used to quantify human milk total GAGs. RESULTS Highest GAG values are present at day 4 (9.3 and 3.8 g/l in preterm and term milk, respectively), followed by a progressive decrease up to day 30 (4.3 and 0.4 g/l). The more remarkable differences are related to the first phases of lactation in which a strong decrease in GAGs was observed between days 4 and 10 (about -73% in term and -50% in preterm newborns). CONCLUSIONS During the first month of lactation, the absolute amount of polysaccharides was constantly and significantly higher in preterm than in term milk, with a similar behavior in the decrease. These data further indicate that human milk GAGs may have an active role in protecting newborns during the first phases of lactation.