To study the effect of different concentrations(50, 100, 200 μg/mL) of Opuntia dillenii Haw. polysaccharides (ODPs) on Toll-like receptor 4(TLR4) and MyD88-dependent signaling pathway, mouse macrophage cells(RAW264.7) were cultured in vitro and treated with ODPs for 24 hours first. Then, the cells were collected and real-time fluorescence quantitative RT-PCR was used to detect the gene expression of TLR4, MyD88, TRAF6 and IKKβ. Western blot was used to detect the expression of TLR4, MyD88, TRAF6 and IKKβ proteins. The results showed that the expression of TLR4, TRAF6 and IKKβ genes and the expression of TLR4, MyD88, and TRAF6 proteins were significantly increased by medium and high doses(100 and 200 μg/mL) of ODPs(P<0.05). They had a good dose-effect relationship. The expression of MyD88 gene was significantly reduced by different concentrations of ODPs(P<0.05). But the increase of IKKβ protein expression was not significant. In summary, ODPs can promote high expression of TLR4 gene and protein and activate the MyD88-dependent pathway of intracellular signal transduction of TLR4 under non-inflammatory conditions, which may be one of the mechanisms that ODPs exert their effects on enhancing immunity.