Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is cytotoxic to most thyroid cancer cell lines including those originating from anaplastic carcinomas, implying TRAIL as a promising therapeutic agent against thyroid cancers. However, signal transduction in TRAIL-mediated apoptosis is not clearly understood. In addition to its well known glycolytic functions, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional protein including its surprising role as a mediator for cell death. In this study, we explored the involvement of GAPDH in TRAIL-mediated thyroid cancer cell death. In FRO cells, S-nitrosylation and nuclear translocation of GAPDH appears to mediate TRAIL-induced cell death at least partially, as evidenced by that pre-treatment with L-NAME, a competitive nitric oxide synthase (NOS) inhibitor partially but significantly attenuated TRAIL-induced apoptosis through the reduction of S-nitrosylation and nuclear translocation of GAPDH. In addition, GAPDH siRNA partially prevented the apoptotic effect of TRAIL, although TRAIL-induced NOS stimulation and production of NO was not attenuated. Furthermore, nuclear localization of GAPDH was observed in another thyroid cancer cell line KTC2, which is also sensitive to TRAIL, but not in those TRAIL insensitive cell lines: ARO, KTC1 and KTC3. These data indicate that NO-mediated S-nitrosylation of GAPDH and subsequent nuclear translocation of GAPDH might function as a mediator of TRAIL-induced cell death in thyroid cancer cells.