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 An in vitro neuroinflammation model was induced by LPS stimulation of BV-2 cells. BV-2 cells were divided into blank (CON), model (LPS), dexamethasone (DEX), and low- and high-dose CG (CG 10 μmol/L, CG 20 μmol/L, respectively) groups. The cell viability in each group was detected by Cell Counting Kit-8 assay, interleukin (IL)-6 and tumor necrosis factor (TNF)-α levels in the supernatant were detected by enzyme-linked immunosorbent assay (ELISA), and nitric oxide levels were detected using the Griess method. LPS was also used to induce neuroinflammation in mice in vivo. The mice were then divided randomly into blank (CON), model (LPS), aspirin, and low- and high-dose CG (CG 5 mg/kg, CG 10 mg/kg, respectively) groups. Pathological changes in the hippocampus were detected by hematoxylin/eosin staining. Serum levels of IL-6 and TNF-α were detected by ELISA, polarization of microglia was detected by immunofluorescence staining, and protein expression levels of Toll-like receptor 4 (TLR4), myeloid differentiation primary response 88 (MyD88), nuclear factor κB (NF-κB, P65) and phosphorylated-NF-κB (p-P65) in the cortex were detected by Western blot. Results CG alone or in combination with LPS in the concentration range of 2.5~160 μmol/L had no significant toxicity in BV-2 cells in vitro, compared with the CON group (P>0.05). IL-6, TNF-α, and NO levels in the cell supernatant were increased in the LPS group compared with the CON group (P<0.01), but were significantly reduced by CG (P<0.05, P<0.01). Hippocampal neurons were arranged loosely and disordered in the LPS group in vivo, compared with the CON group, and nuclear pyknosis was observed. Serum levels of IL-6 and TNF-α were increased (P<0.05, P<0.01). The number of ionized calcium binding adaptor molecule 1 (Iba1) /inducible nitric oxide synthase (iNOS) cells was increased (P<0.01), the number of CD206/Iba1 cells was decreased (P<0.01), and expression levels of TLR4, MyD88, and p-P65 protein in the cortex were increased (P<0.05). Compared with the LPS group, CG improved the pathological damage to the hippocampus and inhibited serum levels of IL-6 and TNF-α (P<0.01). CG also decreased the number of iNOS/Iba1 cells, increased the number of CD206/Iba1 cells (P<0.05,P<0.01), and significantly down-regulated TLR4, MyD88, and p-P65 protein levels in the cortex (P<0.05). Conclusions CG can ameliorate neuroinflammation in mice by suppressing the TLR4/MyD88/NF-κB pathway.