It was reported that the total polysaccharides extracts from Anemarrhenae asphodeloides Bge (Liliaceae, rhizome) could inhibit inflammatory responses in various models. In the present study, the effects of Timosaponin B-II, a purified extract from A. asphodeloidesb, on the expressions of IL-1beta, TNF-alpha and IL-6, the activity of NF-kappaB and the activation of signal pathway related to NF-kappaB were explored in vitro. Timosaponin B-II significantly attenuated increase of these cytokines on both mRNA and protein levels from LPS-stimulated BV2 cells in a dose-dependent manner. The reporter gene assay also showed that the activation of NF-kappaB induced by LPS was inhibited by pre-treatment with Timosaponin B-II. Moreover, western blot results showed that the activation of p38, JNK and P65 had been decreased. These results suggest that both NF-kappaB signal pathway and MAPK pathway were involved in the inhibitory effects of Timosaponin B-II on the expression of pro-inflammatory cytokines.