Interleukin-10-deficient (IL-10^-/-) mice develop an IL-12-mediated intestinal inflammation in the absence of endogenous IL-10. The molecular mechanisms of the dysregulated IL-12 responses in IL-10^-/- mice are poorly understood. In this study, we investigated the role of nuclear factor-κ B (NF-κB) and signal transducers and activators of transcription 3 (STAT3) in lipopolysaccharide (LPS)-induced IL-12p40 gene expression in bone marrow derived-dendritic cells (BMDCs) isolated from wild-type (WT) and IL-10^-/- mice. We report higher IL-12p40 mRNA accumulation and protein secretion in LPS-stimulated BMDCs isolated from IL-10^-/- compared with WT mice. LPS-induced NF-κB signaling is similar in IL-10^-/- and WT BMDCs as measured by IκBα phosphorylation and degradation, RelA phosphorylation and nuclear translocation, and NF-κB transcriptional activity, with no down-regulatory effects of exogenous IL-10. Chromatin immunoprecipitation demonstrated enhanced NF-κB (cRel, RelA) binding to the IL-12p40 promoter in IL-10^-/- but not WT BMDCs. Interestingly, LPS induced STAT3 phosphorylation in WT but not IL-10^-/- BMDCs, a process blocked by IL-10 receptor blocking antibody. Adenoviral gene delivery of a constitutively active STAT3 but not control green fluorescence protein (GFP) virus blocked LPS-induced IL-12p40 gene expression and cRel recruitment to the IL-12p40 promoter. In conclusion, dysregulated LPS-induced IL-12p40 gene expression in IL-10^-/- mice is due to enhanced NF-κB recruitment to the IL-12p40 promoter in the absence of activated STAT3.