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Purpose: Radiotherapy is one of the most important therapeutic options used to treat cancers. Radiation effects can be improved using nanoparticles and chemotherapeutic drugs as radiosensitizing agents. The aim of the present study was to evaluate the effects of folic acidconjugated gold nanoparticles (GNP-F) in combination with doxorubicin (DOX) and x-Ray irradiation in colorectal cancer (CRC) cell line (HT-29). Methods: The cell viability assay (WST-1) was performed to study the cytotoxic effects of different concentrations of DOX and GNP-F after 24 and 48 hours treatments. Then, the effects of the GNP-F, X-Ray irradiation, and DOX drug in single and combined treatments were examined after 24 and 48 hours treatment with effective doses. Likewise, the caspase 3 gene expression ratio and the caspase 3 activity were assessed after 48 h treatment. Moreover, the malondialdehyde (MDA) level was determined in treated and untreated cells. Results: When GNP-F (at a concentration of 70 μM) was combined with X-ray irradiation (2 Gy) and DOX drug, induced more cytotoxic effects compared to the control group. The results of cell viability assay showed that GNP-F + X-Ray in combination with a low concentration of DOX (0.25 x IC50) enhanced the cytotoxic effects of cells compared to related single treatments. Caspase 3 gene expression ratio and caspase 3 activity increased in double and triple combination treatments in comparison with the single groups. Moreover, the MDA level increased in triple combination compared to the single treatments. Conclusion: Our findings confirmed the potential anti-cancer effects of the GNP-F and DOX in combination with X-Ray irradiation in CRC cells. [ABSTRACT FROM AUTHOR]