Gangliosides are complex glycosphingolipids, which exert immune-modulating effects on various cell types. Ganglioside GD₃ and GM₃ are the predominant gangliosides of human breast milk but during the early phase of lactation, the content of GD₃ decreases while GM₃ increases. The biological value of gangliosides in breast milk has yet to be elucidated but when milk is ingested, dietary gangliosides might conceptually affect immune cells, such as dendritic cells (DCs). In this study, we address the in vitro effect of GD₃ and GM₃ on DC effector functionalities. Treatment of bone marrow-derived DCs with GD₃ before lipopolysaccharide-induced maturation decreased the production of interleukin-6 (IL-6), IL-10, IL-12 and tumor necrosis factor-α as well as reduced the alloreactivity in mixed leucocyte reaction (MLR). In contrast, only IL-10 and IL-12 productions were significantly inhibited by GM₃ and the potency of DCs to activate CD4⁺ cells in MLR was unaffected by GM ₃. However, both gangliosides suppressed expression of CD40, CD80, CD86 and major histocompatibility complex class II on DCs. Because GD ₃ overall inhibits DC functionalities more than GM₃, the immune modulating effect of the ganglioside fraction of breast milk might be more prominent in the commencement of lactation during which the milk contains the most GD₃.