Acute alcohol binge results in immunosuppression and impaired production of proinflammatory cytokines, including TNF-alpha. TNF-alpha production is induced by LPS, a TLR4 ligand, and is tightly regulated at various levels of the signaling cascade, including the NF-kappaB transcription factor. Here, we hypothesized that acute alcohol induces TLR4/LPS tolerance via Bcl-3, a nuclear protein and member of the NF-kappaB family. We found that acute alcohol pretreatment resulted in the same attenuating effect as LPS pretreatment on TLR4-induced TNF-alpha production in human monocytes and murine RAW 264.7 macrophages. Acute alcohol-induced Bcl-3 expression and IP studies revealed increased association of Bcl-3 with NF-kappaB p50 homodimers in alcohol-treated macrophages and in mice. ChIP assays revealed increased occupancy of Bcl-3 and p50 at the promoter region of TNF-alpha in alcohol-pretreated cells. To confirm that the Bcl-3-p50 complex regulates transcription/production of TNF-alpha during acute alcohol exposure, we inhibited Bcl-3 expression using a targeted siRNA. Bcl-3 knockdown prevented the alcohol-induced inhibition of TNF-alpha mRNA and protein production. In a mouse model of binge alcohol, an increase in Bcl-3 and a concomitant decrease in TNF-alpha but no change in IL-10 production were found in mice that received alcohol followed by LPS challenge. In summary, our novel data suggest that acute alcohol treatment in vitro and in vivo induces molecular signatures of TLR4/LPS tolerance through the induction of Bcl-3, a negative regulator of TNF-alpha transcription via its association with NF-kappaB p50/p50 dimers.