Objective To explore the effects and mechanism of Calycosin-7-glucoside (CG) on lipopolysaccharide (LPS) induced inflammatory injury in BV-2 cells and in a mouse model of neuroinflammation. Methods In vitro neuroinflammation model was induced by LPS stimulation of BV-2 cells , BV-2 cells were divided into blank (CON) group, model (LPS) group, dexamethasone (DEX) group, low and high dose of Calycosin-7-glucoside (CG 10 μmol/L, CG 20 μmol/L) group. The viability of BV-2 cells was detected by CCK8 assay. The levels of IL-6 and TNF-α in the supernatant of BV-2 cells were detected by ELISA. Griess method was used to measure the level of NO. In vivo, lipopolysaccharide (LPS) was used to induce neuroinflammation in mice,The mice were randomly divided into blank (CON) group, model (LPS) group, aspirin (ASA) group, low and high dose Calycosin-7-glucoside (CG 5 mg/kg, CG 10 mg/kg) groups. Hematoxylin-eosin (HE) staining was used to observe the pathological changes of hippocampus. ELISA was used to measure the serum levels of IL-6 and TNF-α. Immunofluorescence staining was used to observe the polarization of microglia. The protein expressions of TLR4, MyD88, NF-κB (P65) and p-NF-κB (p-P65) in the cortex were detected by Western blot. Results In vitro experiments showed that CG alone or in combination with LPS in the concentration range of 2.5-160 μmol/L had NO significant toxicity to BV-2 cells compared with CON group(P>0.05). The levels of IL-6, TNF-α and NO in the supernatant of BV-2 cells in the LPS group increased(P<0.01). Compared with LPS group, CG significantly decreased the levels of IL-6, TNF-α and NO in the cell supernatant(P<0.05, P<0.01). In vivo experiments showed that compared with the CON group, the hippocampal neurons in the LPS group were arranged loosely and disorderly, and the nucleus showed nuclear pyknosis.. The levels of IL-6 and TNF-α in serum were increased(P<0.05, P<0.01). The number of Iba1+iNOS+ positive cells was increased(P<0.01), the number of Iba1+CD206+ positive cells was decreased(P<0.01), and the expression of TLR4, MyD88 and p-P65 protein was increased in the cortex(P<0.05). Compared with the LPS group, CG improved the pathological damage of hippocampus and inhibited the serum levels of IL-6 and TNF-α(P<0.01). The number of Iba1+iNOS+ positive cells was decreased(P<0.05), the number of Iba1+CD206+ positive cells was increased(P<0.01), and the expression of TLR4, MyD88 and p-P65 protein was significantly down-regulated in the cortex(P<0.05). Conclusion Calycosin-7-glucoside ameliorates neuroinflammation in mice by suppressing the TLR4/MyD88/NF-κB pathway.