Abstract: The aim of this work was to apply response surface methodology (RSM) to model and optimize the accelerated solvent extraction (ASE) technique for extracting the crude polysaccharides (GLP-K) from Gracilaria lemaneiformis. In terms of the yield of polysaccharide, single factor tests and Box-Behnken design response surface method were developed. The structure of the prepared polysaccharides was characterized by Fourier transform infrared (FT-IR) and high performance liquid chromatography (HPLC). And the anti-inflammatory potential of GLP-K in lipopolysaccharide (LPS)-induced RAW264.7 macrophages was also explored. Briefly, the optimal extraction conditions for GLP-K were as follows: 70 °C of extraction temperature, 8.5 min of extraction time, and 4 extraction cycles. Under the condition, the experimental yield of polysaccharide was 9.58%±0.31%. FT-IR showed that the polysaccharide contained uronic acid, and the weight-average molecular weight ranged from 4.4 to 747.1 kDa. GLP-K had no significant cytotoxic effects at or below the concentrations of 1000 μg/mL (P<0.001). Compared with the model group, GLP-K administration group (50, 100, 200, 300, 400, 500 μg/mL) displayed remarkable inhibitory effects on the release level of NO (P<0.001), which decreased by 43.76%~69.47%. This paper enriched the research on the extraction of polysaccharides by accelerated solvent extraction technology, and provided experimental basis for the development and utilization of Gracilaria lemaneiformis polysaccharides.