Scope The relationship between the immune modulatory properties of different mixed linkage β-glucans from plant and microbial origin and their physico-chemical properties is elusive. By the use of murine dendritic cells and multivariate data analysis, this study examines the importance of the physico-chemical characteristics of β-glucans for their capacity to modulate immune responses. Methods and results A total of 23 different β-glucan samples characterised with respect to compositional, molecular, structural and rheological properties were investigated for their immune modulatory properties. Most of the β-glucans showed a decrease in the LPS induced IL-12 production and an increase in the LPS induced IL-10 levels in dendritic cells. When the dendritic cells were stimulated with the probiotic Lactobacillus acidophilus NCFM, the β-glucans up-regulate the IL-12 production but showed no effect on the IL-10 levels. The modulation of the cytokine responses was shown to depend on the β-glucan block structure as expressed by the ratio of cellotriosyl to cellotetraosyl units in the molecule as well as the polymer solubility and aggregation in solution. Conclusion This comparative study reveals that the β-glucan fine structure and in turn the intermolecular organisation in solution are key factors that determine their immune modulatory capacity.