Anaplastic thyroid cancer is among the most aggressive of human cancers, and currently there are few effective treatments for most patients. YM155, first identified as a survivin inhibitor, was highlighted in a high-throughput screen performed by the National Cancer Institute, killing anaplastic thyroid cancer cells in vitro and in vivo. However, there was no association between survivin expression and response to YM155 in clinical trials, and YM155 has been mostly abandoned for development despite favorable pharmacokinetic and toxicity profiles. Currently, alternative mechanisms are being explored for YM155 by a number of groups. In this study, anaplastic thyroid cancer patient samples show overexpression of topoisomerase Top2α compared to benign thyroid samples and to differentiated thyroid cancers. Anaplastic thyroid cancer cell lines that overexpress Top2α are more sensitive to YM155. We created a YM155-resistant cell line, which showed decreased expression of Top2α and is re-sensitized with Top2α overexpression. Molecular modeling predicts binding for YM155 in the Top2α ATP binding site and identifies key amino acids for YM155-Top2α interaction. A Top2α mutant abrogates the effect of YM155, confirming the contribution of Top2α to YM155 mechanism of action. Our results suggest a novel mechanism of action for YM155 and may represent a new therapeutic approach for the treatment of anaplastic thyroid cancer.