Broncho-Vaxom (OM85-BV) is an extract mixture from 8 strains of Gram(+) and Gram(-) bacteria and plays an important role in anti-infection immune response by regulating macrophage activity and cytokine productions. However, the mechanism by which OM85-BV enhances the cytokine expression is still obscure. In this study, we evaluated the effects of OM85-BV on the productions of interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α) in RAW264.7 murine macrophages. Exposure of RAW264.7 cells to 100 μg/mL OM85-BV upregulated the expression of IL-1β, IL-6, and TNF-α at the mRNA and protein levels in a time- and dose-dependent manner. In addition, OM85-BV induced extracellular signal-regulated kinase (ERK) 1/2 and nuclear factor-kappa B (NF-κB) phosphorylation. Pretreatment with U0126 or Bay11-7082, respectively, could decrease IL-1β, IL-6, and TNF-α productions induced by OM85-BV. Application of Toll-like receptor (TLR) 4 or TLR2 small-interfering RNA (siRNA) into RAW264.7 cells could inhibit the productions of cytokines and ERK1/2 and NF-κB phosphorylation induced by OM85-BV. Consistent with this, downregulating either myeloid differentiation factor 88 (MyD88) or TRIF-related adaptor molecule (TRAM) gene with MyD88-siRNA or TRAM-siRNA separately could reduce the productions of cytokines and ERK1/2 and NF-κB phosphorylation induced by OM85-BV. Our study demonstrated that the productions of IL-1β, IL-6, and TNF-α induced by OM85-BV in RAW264.7 cells were through TLR4 and TLR2 signaling pathway-mediated activation of ERK1/2 and NF-κB.