Breastfeeding is recommended by the WHO for the first 6 months of life. It provides health benefits to infants compared to infant formula (IF), including protection against intestinal and respiratory infections in early childhood and a lower risk of metabolic and immune diseases later in life. These differences in health benefits are probably due to the difference in composition between IF and human milk (HM), the latter being more complex and richer in bioactive components. Our hypothesis is that HM health benefits come in part from the HM microbiota -a paucimicrobial ecosystem that presents a great bacterial diversity- through a role on gut immune homeostasis. This study aimed to better understand the immunomodulatory role of HM bacteria. To this end, bacteria were isolated from healthy breast milks and screened for their immunomodulatory potential on two different cellular models in order to select candidates with probiotic interest. The collection of 28 breast milks at the Rennes Sud University Hospital allowed the constitution of a strain library of 1164 isolates with 29 genera and 56 different species. From this library, 88 isolates selected for their prevalence in HM microbiota were first screened on PBMC (blood mononuclear cells) for the production of IL-10 and TNF-alpha cytokines. Among them, 28 isolates exhibiting interesting immunomodulatory properties on PBMC model underwent a second screening on a quadricellular model of intestinal epithelium including enterocytes (Caco2), caliciform cells (HT 29-MTX), M cells (differentiated Caco2) and macrophages (THP-1). This study showed the species- and strain-dependence of HM bacteria immunomodulatory potential and revealed interesting properties of HM bacteria in the context of infant immune system maturation. This study is part of the PROLIFIC project funded by the Régions Bretagne and Pays de la Loire and by the BBA MilkValley industrial consortium.