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OBJECTIVE: Thyroid cancer is one of the most prevalent cancers of the endocrine system. Allium jesdianum from the Liliaceae family is suggested as an herbal remedy for numerous human disorders. Due to the need to identify a new treatment strategy for thyroid cancer and due to the many therapeutic properties of Allium jesdianum, beneficial biologically active compounds, and the anti-cancer activity of these compounds, this study aimed to investigate the effect of this herb extract on thyroid cancer cells (B-CPAP and Thr.C1-PI 33). MATERIALS AND METHODS: The hydro-alcoholic extract of Allium jesdianum was prepared. The effects of the extract on the viability, apoptosis, and nitric oxide production of human thyroid cancer cell lines were investigated. Real-time PCR is used to analyze the apoptosis-related genes expression pattern. RESULTS: MTT assay showed that the extract had cytotoxic activity against both cell lines. In addition, treatment of cells with various concentrations of the extract caused the release of lactate dehydrogenase and induction of apoptosis cell death. NO production was decreased by the treatments in a concentration- and time-dependent manner. The extract stimulated apoptosis cell death by decreasing the expression of Bcl-2 and increasing the expression of Bax, p53, and Caspase 3. CONCLUSIONS: The extract possesses potent cytotoxic activity and anti-cancer properties which are possibly due to the principal bioactive phytochemical composites existing in this plant. These results can be used to develop new drugs for thyroid cancer treatment.