Toll-like receptor 4 (TLR4) is expressed in head and neck squamous cell carcinoma (HNSCC) cells and is associated with HNSCC cancer progression. Rapamycin has been proven to be efficient for the treatment of HNSCC in vivo, yet the mechanism is not understood and rapamycin demonstrates little effect in vitro. In the present study, the HNSCC cell lines CAL27 and SCC4 were pre-treated with rapamycin then stimulated with a TLR4 ligand lipopolysaccharide (LPS). Cell proliferation, migration, invasion, resistance to TRAIL-induced apoptosis, cytokine production, NF-κB and p65 activation were determined. The results indicated that LPS significantly stimulated HNSCC cell proliferation, cytokine production, migration, invasion and resistance to apoptosis induced by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL). Pretreatment with rapamycin significantly attenuated LPS-induced pro-oncogenic effects by inhibiting the activation of NF-κB by LPS. siRNA knockdown of TLR4 in HNSCC cells demonstrated that rapamycin attenuated LPS-induced pro-oncogenic effects via TLR4. Hence, this study suggests rapamycin may be efficient for the treatment of HNSCC by attenuating TLR4-induced pro-oncogenic effects.