Background: Adrenocortical carcinoma (ACC), is an uncommon and highly aggressive malignant tumor with endocrine functions, typically associated with adverse clinical outcomes. Mitotane is the only drug currently available; however, it has poor efficacy and several side effects. Abiraterone, a CYP17A1 inhibitor, has been widely used to treat desmoplasia-resistant prostate cancer; however, its antitumor activity in ACC remains unknown. Objective: To examine the potential inhibitory effects of abiraterone acetate on the malignant progression of ACC. Methods: The ACC cell line NCI-H295R was used to evaluate the cytotoxic effects of abiraterone acetate. We determined the IC50 using the CCK8 assay. Apoptotic activity and cell cycle dynamics were evaluated through flow cytometry. Cell growth was assessed with a colony formation assay. Invasive and migratory abilities were assessed using a Transwell assay. Next-generation sequencing (NGS) was followed by drug-regulated gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses. EFNA3 expression was manipulated using plasmid overexpression and lentiviral knockdown. Results: Abiraterone acetate inhibited ACC cell viability, migration, and invasion and induced apoptosis and G2/M phase blockade in ACC cells. Bioinformatic analysis revealed that EFNA3, a critical determinant in the regulation of cell migration and invasion, was significantly downregulated in abiraterone-treated ACC cells. EFNA3 overexpression partially reversed the inhibitory effects of abiraterone on cell migration and invasion, whereas EFNA3 knockdown abrogated these effects. Conclusion: Abiraterone acetate inhibited the malignant behavior of ACC in vitro. EFNA3 mediates the anti-invasion and anti-metastatic effects of abiraterone acetate in ACC cells.