RNA interference has been extensively used to knock-down the translation of certain genes. Toll-like receptor 4 (TLR4) produced by macrophages can be activated in response to endotoxin stimulation. This study used the RNA interference technique to evaluate the roles of TLR4 in lipopolysaccharide (LPS)-stimulated activation of macrophages from the aspects of cytokine production, chemotaxis, phagocytosis, and oxidative ability. Exposure of macrophages to 1, 25, 50, 100 ng/mL LPS for 1, 6, and 24 h did not affect cell viability. Meanwhile, treatment with 100 ng/mL LPS induced interleukin (IL)-1beta protein and mRNA syntheses in a time-dependent manner. Application of TLR4 small interference (si)RNA into macrophages decreased the levels of this receptor, and simultaneously ameliorated LPS-induced IL-1beta and IL-6 mRNA production. Transwell analysis showed that LPS increased chemotactic activity of macrophages, but application of TLR4 siRNA reduced such an effect. Phagocytic activities of macrophages were significantly augmented following LPS treatment. However, knocking-down the translation of TLR4 mRNA using RNA interference lowered the LPS-enhanced phagocytic activity. Analysis of flow cytometry revealed that LPS increased oxidative ability of macrophages, but TLR4 siRNA inhibited such development. This study used RNA interference techniques to show that TLR4 can mediate LPS-induced macrophage activations of IL-1beta and IL-6 gene expression, chemotaxis, phagocytosis, and oxidative ability.