To investigate the anti-inflammatory activity of Ganoderma lucidum polysaccharides (GLP) in vitro by different extraction methods in RAW264.7 macrophages stimulated by LPS. In this experiment， the extraction of GLP was carried out by hot water， multiple enzyme， fermentation with L. plantarum combined with hot water， fermentation with L. plantarum combined with multiple enzyme. In the process of in vitro experiments， after stimulation of GLP for 24 h， CCK8 assay was used to detect the effect of GLP on the proliferation of RAW264.7 macrophages. The mRNA expression levels of TNF-α， IL-6， IL-10 and iNOS in RAW264.7 macrophages was observed by qRT-PCR. The expression of phosphorylated JNK (P-JNK) and phosphorylated P38 MAPK (P-P38 MAPK) proteins was detected by Western blot. Results indicated that The survival rates of RAW264.7 cells treated with four types of GLP under mass concentration of 200 μg/mL were above 93%. Compared with LPS， the four types of GLP exhibited a more significant (P＜0.05) influence on the mRNA expression levels of TNF-α， IL-6， IL-10 and iNOS in RAW264.7 macrophages. GLP of fermentation with L. plantarum combined with hot water extraction had a more significant(P＜0.05) effect than GLP extracted by hot water alone on the mRNA expression levels of TNF-α， IL-6， IL-10. GLP of fermentation with L. plantarum combined with multiple enzyme extraction had a more significant (P＜0.05) effect than GLP extracted by other three extraction methods on the mRNA expression levels of iNOS. GLP of fermentation with L. plantarum had a more significant (P＜0.05) effect than GLP extracted solely by hot water and multiple enzyme on the expression levels of JNK/P38 MAPK signal pathway relatived proteins in RAW264.7 cells. This study demonstrated that GLP possesses significant anti-inflammatory activity in vitro， and the extraction methods greatly influenced the anti-inflammatory activity of GLP in vitro. GLP of fermentation with L. plantarum showed a more significant effect than GLP extracted by hot water and multiple enzyme in terms of anti-inflammatory activity.