Objective To investigate the inhibitory effect of total ginkgo flavonoids (TFG) on lipopolysaccharide (LPS)-induced inflammatory response in microglial cells. Methods The microglial cell line BV-2 was activated by LPS to establish a model of neuroinflammation, and then the cells were treated with different concentrations of TFG (0, 40, 80, and 160 mg/ml, respectively). CCK-8 assay was used to measure cell viability; the cells pretreated with TFG at a concentration of 80 mg/ml were selected and an inverted phase-contrast microscope was used to observe the morphological changes of microglial cells. ELISA was used to measure the levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α); Western blot was used to measure the protein expression of Toll-like receptor 4 (TLR4), inhibitor kappa B (IκB-α), and nuclear factor-κB (NF-κB)/p65. Results The low-dose TFG groups (40 and 80 mg/ml) had no significant change in the activity of microglial cells (P>0.05), while the high-dose TFG group (120 and 160 mg/ml) had significant inhibition of the activity of microglial cells (P<0.05). Compared with the LPS group, the TFG+LPS group had significant inhibition of LPS-induced morphological changes of microglial cells and significantly lower levels of cytokines IL-1β, IL-6, and TNF-α (P<0.05), as well as significantly lower protein expression of TLR4 (P<0.05), a significantly higher expression of IκB-α in cytoplasm (P<0.05), and significant inhibition of nuclear translocation of NF-κB/p65 (P<0.05). Conclusions TFG has a good inhibitory effect on LPS-induced inflammatory response in microglial cells and thus has a certain therapeutic effect on neurodegenerative diseases characterized by neuroinflammation.