As one of the most important components of Panax ginseng, ginsenoside Rg1 has certain anti-aging effects, improving the activity of learning and memory. Studies have showed that ginsenoside Rg1 improves the memory impairment associated with Alzheimer’s disease (AD). In this study, the effects of ginsenoside Rg1 were investigated through the activity of toll-like receptor (TLR) 3, TLR4 and their signaling transduction pathways in amyloid β peptide 25–35 (Aβ25–35) induced AD cell model. Thus we investigated several critical components of the TLR pathway. The neuroglial cell line NG108-15 was stimulated with or without Aβ25–35, while different concentrations of ginsenoside Rg1 were administered. After 24 h, tumor necrosis factor-α (TNF-α), interferon-β (IFN-β) in cell supernatant and inducible nitric oxide synthase (iNOS) in cell lysate supernatant were measured with enzyme-linked immunosorbent assays (ELISAs). The mRNA and protein expression of TLR3, TLR4, nuclear factor kappa B (NF-κB) and tumor necrosis factor receptor-associated factor-6 (TRAF-6) were detected by real-time PCR and western blot methods, respectively. The experimental results showed that Aβ25–35 could markedly raise the level of TNF-α, IFN-β and iNOS, and increase the expressions of mRNA and TLR3, TLR4, NF-κB and TRAF-6 protein in the NG108-15 cells. At the same time, the ginsenoside Rg1 significantly reduced the expressions of proteins and mRNA of TLR3, TLR4, NF-κB and TRAF-6, and down-regulated the levels of TNF-α, IFN-β of cell supernatant and iNOS of cell lysate supernatant in a concentration-dependent manner. In conclusion, ginsenoside Rg1 has good activity for suppressing the signaling transduction pathway of TLR3 and TLR4, and decreasing the inflammation factors induced by Aβ25–35 in NG108-15 cells, and this may be the mechanism of ginsenoside Rg1 action in AD treatment, but more studies are needed to identify its specificity.