Abstract: To improve the extraction efficiency of Tremella fuciformis polysaccharides (TP) and to investigate their structural characteristics and anti-inflammatory activity, dynamic high-pressure microfluidization (DHPM) was employed as an auxiliary extraction technique, and the extraction parameters were optimized using single-factor experiments and Box-Behnken response surface methodology. TP was structurally characterized by high-performance gel permeation chromatography (HPGPC), ion chromatography (IC), and Fourier transform infrared spectroscopy (FT-IR), and its anti-inflammatory activity was evaluated in a lipopolysaccharide (LPS)-induced RAW264.7 macrophage inflammation model. Results showed that a regression model with microfluidization pressure, solid–liquid ratio, and extraction time as independent variables exhibited good fitness. The optimal extraction conditions were determined to be a microfluidization pressure of 140 MPa, a solid-liquid ratio of 1:40 (g/mL), and an extraction time of 90 min, under which the TP yield reached 36.01%. Chemical composition analysis revealed that TP contained 86.12% total sugars and 33.11% uronic acids, with no detectable free proteins or monosaccharides, indicating that TP was a highly purified acidic polysaccharide. Structural analyses further showed that TP had an average molecular weight of 568385 Da and was composed of xylose, fucose, mannose, glucuronic acid, galactose, and glucose, in which the relatively high proportion of glucuronic acid confirmed its acidic characteristics. FT-IR spectra demonstrated that TP was mainly composed of β-pyranose-linked polysaccharide chains. In vitro experiments indicated that TP exhibited no cytotoxicity within the concentration range of 100~200 μg/mL and significantly reduced the LPS-induced secretion of NO, TNF-α, and IL-1β, while increasing the production of the anti-inflammatory cytokine IL-10 (P<0.05), demonstrating its notable anti-inflammatory potential. In conclusion, TP prepared by DHPM exhibits clear structural features, high purity, and remarkable anti-inflammatory activity, highlighting its potential as a natural functional ingredient for inflammation-related applications.