The molecular architecture and physicochemical traits of β-glucans vary among their sources, potentially influencing their biological activity. In this study, we analyzed the molecular configurations of four β-glucans sourced from yeast and euglena, and assessed their impact on immune regulation using RAW264.7 macrophages activated by lipopolysaccharide (LPS). The findings revealed that yeast-derived β-glucans have a smaller particle size than those from euglena, yet the yeast β-glucans possess a higher molecular weight. The ratio of glycoside bonds indicated that yeast β-glucans possess side chains with both β-1,3 and β-1,6 glycosidic linkages. β-Glucans inhibited LPS-induced release of NO, ROS, TNF-α, and IL-6 in macrophages, enhanced cell phagocytic ability, and improved cell proliferation rate. Samples from different sources and doses exhibited different levels of immune activity. Among them, yeast β-glucans performed better in enhancing cell proliferation ability and inhibiting the release of NO, ROS, IL-6, and TNF-α by macrophages, while euglena β-glucan treatment had higher phagocytic ability. It was hypothesized that β-glucans with smaller particle size, higher molecular weight, and complex branched structures would probably show stronger immunoregulatory activity. PRACTICAL APPLICATIONS: The results of this study provided potential criteria for selecting β-glucans as immunoregulatory supplements and helped to elucidate the immunomodulatory mechanisms of β-glucans.