Recently, we reported that silibinin inhibits primary lung tumor growth and progression in mice and down-regulates inducible nitric oxide synthase (iNOS) expression in tumors; however, the mechanisms of silibinin action are largely not understood. Also, the activation of signaling pathways inducing various transcription factors are associated with lung carcinogenesis and their inhibition could be an effective strategy to prevent and/or treat lung cancer. Herein, we used human lung epithelial carcinoma A549 cells to explore the potential mechanisms and observed strong iNOS expression by cytokine mixture (containing 100 units/mL IFN-gamma + 0.5 ng/mL interleukin-1beta + 10 ng/mL tumor necrosis factor-alpha). We also examined the cytokine mixture-activated signaling cascades, which could potentially up-regulate iNOS expression, and then examined the effect of silibinin (50-200 mumol/L) on these signaling cascades. Silibinin treatment inhibited, albeit to different extent, the cytokine mixture-induced activation of signal transducer and activator of transcription 1 (Tyr(701)), signal transducer and activator of transcription 3 (Tyr(705)), activator protein-1 family of transcription factors, and nuclear factor-kappaB. The results for activator protein-1 were correlated with the decreased nuclear levels of phosphorylated c-Jun, c-Jun, JunB, JunD, phosphorylated c-Fos, and c-Fos. Further, silibinin also strongly decreased cytokine mixture-induced phosphorylation of extracellular signal-regulated kinase 1/2 but only marginally affected JNK1/2 phosphorylation. Silibinin treatment also decreased constitutive p38 phosphorylation in the presence or absence of cytokine mixture. Downstream of these pathways, silibinin strongly decreased cytokine mixture-induced expression of hypoxia-inducible factor-1alpha without any considerable effect on Akt activation. Cytokine mixture-induced iNOS expression was completely inhibited by silibinin. Overall, these results suggest that silibinin could target multiple cytokine-induced signaling pathways to down-regulate iNOS expression in lung cancer cells and that could contribute to its overall cancer preventive efficacy against lung tumorigenesis.