Immunomodulatory oligosaccharides found on helminths also are found in human milk, and both helminths and milk have been shown to be immunosuppressive. We have been examining the immunomodulatory capabilities of two oligosaccharides expressed in milk and on helminth parasites, lacto-N-fucopentaose III and lacto-N-neotetraose (LNnT). In an attempt to dissect mechanisms that lead to Th2 polarization and immune suppression, we examined the early response in mice to the glycoconjugate LNnT-Dextran (LNnT-Dex). We found that injection of LNnT-Dex expanded a cell population, phenotypically defined as Gr1+/CD11b+/F4/80+, as early as 2 h after injection. Examination of spontaneous cytokine production showed that this Gr1+/F4/80+ population of cells spontaneously produced low levels of proinflammatory cytokines, but higher levels of IL-10 and TGF-β ex vivo, compared to peritoneal cells from mice injected with Dex. Gr1+ cells adoptively suppressed naive CD4+ T cell proliferation in vitro in response to anti-CD3/CD28 Ab stimulation. Suppression of naive CD4+ cells involved cell contact and was dependent on IFN-γ and NO, with a discrete role played by IL-10. Coculture of naive CD4+ T cells with Gr1+ suppressor cells did not lead to CD4+ T cell apoptosis, although it did imprint on naive CD4+ T cells a response characterized by lower levels of IFN-γ, coincident with increased IL-13 production. Our results suggest that both human milk and helminth parasites may share a ligand-specific mechanism involved in the generation of anti-inflammatory mediators that suppress Th1-type and inflammatory responses.