This study comprehensively explores the relationship between the structure of carboxymethyl-pachymaran (CMP) and its diverse biological activities, including immunomodulation, anti-inflammatory effects, tumor cell proliferation inhibition, and antioxidant activity. By adjusting preparation parameters, highly purified CMP samples with varying degrees of substitution (DS) and molecular weights (Mw) were successfully obtained. The results indicate that CMP, composed primarily of β-D-glucan, exhibits different levels of activity depending on its structural characteristics. In terms of immunomodulation, CMP with medium Mw demonstrates the strongest activity, while CMP with a high DS promotes nitric oxide (NO) synthesis most effectively, and a moderate DS supports optimal tumor necrosis factor-alpha (TNF-α) synthesis. For anti-inflammatory activity, CMP with a moderate DS effectively inhibits NO production, while a low DS is most effective against TNF-α inhibition. Larger Mw consistently enhances the inhibitory effects on NO and TNF-α synthesis. Dectin-1 is identified as one of the receptors mediating CMP's immunomodulatory effects. Furthermore, CMP with a moderate DS and high Mw exhibit superior performance in inhibiting HepG-2 cell proliferation and scavenging DPPH free radicals, respectively. This study enriches understanding of β-glucan's structure-activity relationship and lays a theoretical foundation for its medical and healthcare applications.