Grifola frondosa (Maitake) is traditionally valued for its health benefits, with polysaccharides being key bioactive components. This paper investigates a specific subfraction, Fraction D (GFP-D), evaluating its clinical effects and mechanisms in immune enhancement, adjunctive anti-tumor activity, and regulation of glucose/lipid metabolism. Three clinical trials were conducted. In an immune study, 120 healthy volunteers (CD4+ T cell count 500–1000 cells/μL) received 150 mg/day GFP-D for 8 weeks, resulting in significant increases in CD4+ T cells (from 632 ± 95 to 812 ± 108 cells/μL, 28.5% increase, within the physiological activation range), CD4+/CD8+ ratio, NK cell activity, IL-2, and IFN-γ (all P < 0.001 vs. placebo). An anti-tumor study with 80 advanced cancer patients (stratified by age, tumor stage, and histotype) showed that adding 1000 mg/day GFP-D to chemotherapy improved objective response rate (52.5% vs. 30.0%, P = 0.036, 95% CI: 1.02–3.87), one-year progression-free survival (55.8% vs. 33.3%, P = 0.022), and preserved immune parameters versus chemotherapy alone. A metabolic study in 90 type 2 diabetes patients found that 400 mg/day GFP-D for 12 weeks significantly lowered fasting glucose, HbA1c, total cholesterol, triglycerides, and LDL-C, while raising HDL-C (from 1.0 ± 0.2 to 1.2 ± 0.2 mmol/L, 20% increase, supported by increased AMPK phosphorylation). Mechanistically, immune enhancement involves macrophage/dendritic cell activation via Dectin-1/TLR4 receptors (confirmed by increased receptor expression and downstream signaling molecules), promoting cytokine-driven T/NK cell responses. Anti-tumor effects stem from immunomodulation, direct induction of cancer cell apoptosis (via mitochondrial/caspase pathways, verified by increased Bax/Bcl-2 ratio and caspase-3 activation), and angiogenesis inhibition by downregulating VEGF. Metabolic benefits are linked to AMPK pathway activation in liver/muscle (confirmed by increased p-AMPK/AMPK ratio), enhancing glucose uptake and inhibiting gluconeogenesis/lipogenesis, alongside modulation of gut microbiota (increased Bifidobacterium and Lactobacillus abundance). All trials reported no severe adverse events related to GFP-D; liver/kidney function parameters (ALT, AST, creatinine, urea nitrogen) remained within normal ranges throughout the intervention. Collectively, GFP-D emerges as a multi-functional bioactive agent with substantial therapeutic potential.