Background/Aims: The aim of the present study was to identify and quantify the polyamine levels in human milk obtained from different countries and through different modes of delivery, and to investigate their association with breast milk microbes. Methods: Mature breast milk samples were obtained from 78 healthy mothers after 1 month of lactation from 4 different geographical locations: Finland, Spain (Europe); South Africa (Africa); and China (Asia). Polyamines were determined using HPLC after dansyl derivatization and milk microbiota was obtained by 16S rRNA gene sequencing. Results: The mean values of polyamines in breast milk were 70.0, 424.2, and 610.0 nmol/dL for putrescine, spermidine and spermine, respectively, and 1,170.9 nmol/dL of total polyamines. The levels of putrescine were significantly higher in Spain (p < 0.05) and spermidine levels were significantly higher in Finland (p < 0.05) compared with other countries. Cesarean delivery had an impact on polyamine levels and it was related to an increase in the putrescine concentration being significant in Spanish samples (p < 0.01). Furthermore, putrescine levels were correlated positively with Gammaproteobacteria (r = 0.46, p < 0.001), especially with Pseudomonas fragi (r = 0.40, p < 0.001). Conclusions: The results demonstrate significant effect of geographical variations in human milk polyamine concentrations, being correlated with human milk microbiota composition. These differences may have an impact on infant development during lactation.