Understanding the molecular pathways perturbed in smokeless tobacco- (ST) associated head and neck squamous cell carcinoma (HNSCC) is critical for identifying novel complementary agents for effective disease management. Activation of nuclear factor-kappaB (NF-κB) and cyclooxygenase-2 (COX-2) was reported in ST-associated HNSCC by us [Sawhney,M. et al. (2007) Expression of NF-kappaB parallels COX-2 expression in oral precancer and cancer: association with smokeless tobacco. Int. J. Cancer, 120, 2545-2556]. In search of novel agents for treatment of HNSCC, we investigated the potential of guggulsterone (GS), (4,17(20)-pregnadiene-3,16-dione), a biosafe nutraceutical, in inhibiting ST- and nicotine-induced activation of NF-κB and signal transducer and activator of transcription (STAT) 3 pathways in HNSCC cells. GS inhibited the activation of NF-κB and STAT3 proteins in head and neck cancer cells. This inhibition of NF-κB by GS resulted from decreased phosphorylation and degradation of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha the inhibitory subunit of NF-κB. Importantly, treatment of HNSCC cells with GS abrogated both ST- and nicotine-induced nuclear activation of NF-κB and pSTAT3 proteins and their downstream targets COX-2 and vascular endothelial growth factor. Furthermore, GS treatment decreased the levels of ST- and nicotine-induced secreted interleukin-6 in culture media of HNSCC cells. In conclusion, our findings demonstrated that GS treatment abrogates the effects of ST and nicotine on activation of NF-κB and STAT3 pathways in HNSCC cells that contribute to inflammatory and angiogenic responses as well as its progression and metastasis. These findings provide a biologic rationale for further clinical investigation of GS as an effective complementary agent for inhibiting ST-induced head and neck cancer.