Abstract Purpose: Adrenocortical carcinoma (ACC) is a rare and aggressive cancer, and no current effective therapy is available for locally advanced and metastatic ACC. Drug repurposing is an emerging approach for identifying new indications for existing drugs, especially for rare cancers such as ACC. The objective of this study was to use quantitative high-throughput screening to identify agents with antineoplastic activity against ACC. Experimental Design: A screening of 4,292 compounds was performed on three ACC cell lines: BD140A, SW-13, and NCI-H295R. Results: Twenty-one active compounds were identified, with an efficacy of >80% in all three cell lines. Of these, niclosamide showed higher efficacy and lower IC50 than established anti-ACC drugs. We then validated niclosamide-inhibited cellular proliferation in all three ACC cell lines. Next, we investigated the mechanism by which niclosamide inhibited ACC cell proliferation, and found that it induced caspase-dependent apoptosis and G1 cell-cycle arrest. Niclosamide also decreased cellular migration and reduced the level of mediators of epithelial-to-mesenchymal transition, such as N-cadherin and vimentin. Furthermore, niclosamide treatment resulted in decreased expression of β-catenin. We also evaluated the effect of niclosamide on energy metabolism in ACC cell lines and found it resulted in mitochondrial uncoupling. Niclosamide treatment inhibited ACC tumor growth with no observed toxicity in mice in vivo. Conclusions: Our findings suggest that niclosamide has anti-ACC activity through its inhibition of multiple altered cellular pathways and cellular metabolism in ACC. Our results provide a preclinical rationale for evaluating niclosamide therapy in a clinical trial for ACC. Clin Cancer Res; 22(14); 3458–66. ©2016 AACR.