Activated mast cells play an important role in the development and maintenance of chronic inflammation by releasing proinflammatory cytokines such as Tumor necrosis factor alpha (TNFalpha). TNFalpha is a key mediator of immune and inflammatory responses as it controls the expression of inflammatory genes network and its overproduction contributes significantly to the pathological complications observed in many inflammatory diseases. We have previously shown that thymoquinone (TQ), which has broad anti-inflammatory activities, attenuates allergic inflammation in mice. In the present study, we investigated the effect of TQ on LPS-induced TNFalpha production in the rat basophil cell line, RBL-2H3. Stimulation of RBL-2H3 cells with LPS markedly increased TNFalpha production. TQ treatment significantly inhibited LPS-induced TNFalpha mRNA expression and protein production. To understand the mechanism by which TQ inhibited TNFalpha production, we examined its effects on activation of NF-kappaB transcription factor, which has been shown to be involved in regulating TNFalpha responses. LPS activated the NF-kappaB pathway, resulting in accumulation of NF-kappaB p65 and p50 subunits in the nucleus and activation of TNFalpha promoter. TQ administration to LPS-stimulated cells did not noticeably alter NF-kappaB cytosolic activation or nuclear expression as demonstrated by western blot analysis. Instead, TQ significantly increased the amount of the repressive NF-kappaB p50 homodimer, and simultaneously decreased the amount of transactivating NF-kappaB p65:p50 heterodimer, bound to the TNFalpha promoter as revealed by electrophoretic mobility shift and chromatin immunoprecipitation assays. Transient transfection of RBL-2H3 cells with TNFalpha promoter-driven luciferase gene constructs demonstrated that one of the three NF-kappaB binding sites in the TNFalpha promoter, the kappaB3 site, played a major role in the induction of TNFalpha promoter-driven luciferase gene expression by LPS, as well as in mediating the inhibitory effects of TQ on TNFalpha production, as TQ had minimal effect on the TNFalpha promoter-luciferase construct that lacks the kappaB3 site. Together, these results suggest that TQ attenuates the proinflammatory response in LPS-stimulated mast cells by modulating nuclear transactivation of NF-kappaB and TNFalpha production.