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Although numerous polysaccharides have demonstrated potential immunostimulatory activities in in vitro models, only a few of them successfully stimulate the immune system in vivo. In order to explore the possible reasons for the activity loss of polysaccharides in in vivo models, the immunostimulatory activities in vitro and in vivo and the digestion behavior of a polysaccharide from Cyclina sinensis (CSP) were investigated in the present study. CSP showed obvious immunostimulatory activity in a RAW 264.7 cell model. In in vitro experiment, CSP did not exhibit cytotoxicity at concentrations of ≤10 µg/ml, and significantly increased NO production at concentrations of 0.4–10 µg/ml, suggesting CSP processes immunostimulatory activity in vitro. Further investigation using simulated digestion model indicated that CSP could bind with the protein in the digestive fluids to form precipitate in both the stomach and small intestine, and it could be seriously degraded by amylase during the digestion in the small intestine. Furthermore, the in vivo immunostimulatory activity evaluation demonstrated CSP had no effect on immunosuppressed mice as indicated by the body weight, thymus and spleen indexes, and TNF‐α, IL‐1β, IL‐6, and IL‐10 mRNA expression. Thus, the present study indicates that the degradation and precipitation of CSP in the digestive tract are the possible reasons for the activity loss of CSP after digestion. Practical Application: Cyclina sinensis is the common aquatic shellfish in China and plays an important role in the marine aquaculture industry. Cyclina sinensis polysaccharide (CSP) is the main active component of C. sinensis. The structure characterization and immunostimulatory activity of a purified fraction of CSP (CSP‐1) and the effect of digestion on CSP and its immunostimulatory activity were studied. The result of this study promotes the understanding of the nutritional function effects and provides a scientific reference for the rational development and high‐value utilization of C. sinensis. [ABSTRACT FROM AUTHOR]