Anaplastic thyroid cancer (ATC) is the most aggressive thyroid cancer. Current approaches including surgery, chemotherapy and therapeutic drugs provide limited benefit for ATC patients. Diallyl trisulfide (DATS), a garlic-derived organosulfur compound, has been documented for promising anti-cancer effect in various carcinoma. However, it is remained to uncover the role of DATS in ATC tumorigenesis. DATS treatment at 12.5, 25 and 50 μM decreased the viability of ATC cell line 8505C cells determined by SRB, colony formation and Hoechst/PI double staining assays. DATS inhibited the proliferation of 8505C cells both in a dose- and time-dependent manner. Furthermore, DATS induced a G2/M cell cycle arrest which lead to a caspase-dependent mitochondrial apoptosis evidenced by PI-cytometry, Annexin V/PI staining and western blot. The phosphorylation of H2A.X, which is a DNA damage marker, was induced by DATS both in a dose- and time-dependent manner. Moreover, the long tails examined by comet assay further confirmed that DATS induced DNA damage in 8505C cells. The phosphorylation of ATM but not ATR was upregulated under the stimulation with DATS, which then resulted into the activation of target genes Chk1 and Chk2. Further results showed that DATS-induced DNA damage in 8505C cells was ROS-dependent but independent of DNA conformational changes. KU-55933, an inhibitor specific to ATM, reversed the phosphorylation of H2A.X and the cleavage of PARP caused by DATS. Taken altogether, our findings demonstrated that DATS induced an ATM-mediated DNA damage in ATC cells 8505C, which shed a light on the development of novel therapeutic agents for ATC treatment.