Background: Immunomodulatory proteins in human milk (HM) can shape infant immune development. However, strategies to modulate their levels are currently unknown. This study investigated whether maternal prebiotic supplementation alters the levels of immunomodulatory proteins in HM. Methods: The study was nested within the SYMBA double-blind randomised controlled trial (ACTRN12615001075572), which is investigating the effects of maternal prebiotic (scGOS/lcFOS) supplementation from <21 weeks gestation during pregnancy until 6-months postnatal during lactation on child allergic disease risk. Mother-child dyads receiving prebiotics (n=46) or placebo (n=54) were included in this study. We measured the levels of 24 immunomodulatory proteins in HM collected at 2, 4 and 6 months. Results: Cluster analysis showed that the overall immunomodulatory protein composition of milk samples from both groups was similar. Prebiotic-supplemented women had decreased levels of TGF-β1 at 2 months, IgG1 at 4 months and sCD14 at 6 months (p=0.03, 0.03 and 0.04, respectively). IL-5 levels were increased in the prebiotic group compared to the control group at 4 and 6 months (p=0.02 and 0.04, respectively). After adjustment for multiple comparisons, the statistical differences between the prebiotic and placebo groups were no longer present (p>0.05). Conclusion: This study is the first to comprehensively trial scGOS/lcFOS intake during pregnancy and indicate potentially altered levels of immunomodulatory proteins in HM. Future research will elucidate the significance of change in milk content in immunomodulatory compounds in the prevention of allergy risk (and beyond), filling a major gap in dietary advice to pregnant and/or lactating women.