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Objective(s): Taking advantage of high atomic number of gold nanoparticles (GNPs) in radiation dose absorbing, many in vitro and in vivo studies have been carried out on using them as radio-sensitizer. In spite of noticeable dose enhancement by GNPs at keV energies, using this energy range for radiotherapy of deep-seated tumors is outdated. The aim of the present work was to examine the effect of GNPs on radio-sensitivity of HT-29 cells in combination with 18 MV X-rays.Materials and Methods: GNPs were synthesized using a seed-growth method and characterized by transmission electron microscopy (TEM) for size and morphology. Cytotoxicity effect of the GNPs as well as amount of uptake into the HT-29 cell line was assessed. Irradiation was done by 18 MV photons. Immuno-fluorescent imaging of γ-H2AX foci and clonogenic assay were conducted to find out the effect of the GNPs on radio-sensitivity of the cells.Results: The size of GNPs was about 24 nm with a spherical-like shape. Treatment of the cells with the GNPs induced insignificant inhibition in cell growth. Cellular uptake reaches a maximum after 12 h incubation with GNPs. Stained γ-H2AX foci showed a significant difference in number and intensity for GNPs treated cells compared to only irradiated one. Moreover, colony formation assay proved an impressive decrease in the number of colonies for the irradiated+GNPs group rather than the other one. By fitting the survival fraction data on the linear-quadratic model, sensitization enhancement factor (SER) of 1.25 was achieved.Conclusion: Although theoretical studies predicted negligible radio-enhancement factor for GNPs at high megavoltage energies, present results show the potential of GNPs for possible gold nanoparticle-aided radiation therapy (GNRT) even for high MV photons.