Extrachromosomal circular DNAs (eccDNAs) are prevalent in cancer genomes and emerge as a class of crucial yet less characterized oncogenic drivers. However, the structure, composition, genome-wide frequency, and contribution of eccDNAs in hepatocellular carcinoma (HCC), one of the most fatal and prevalent cancers, remain unexplored. In this study, we provide a comprehensive characterization of eccDNAs in human HCC and demonstrate an oncogenic role of microRNA-17-92-containing eccDNAs in tumor progression.Using the Circle-sequencing method, we identify and characterize more than 230,000 eccDNAs from four paired samples of HCC tumor and adjacent non-tumor liver tissues. EccDNAs are highly enriched in HCC tumors, preferentially originate from certain chromosomal hotspots, and are correlated with differential gene expression. Particularly, a series of eccDNAs carrying the microRNA-17-92 cluster are validated by outward PCR and sanger sequencing. Quantitative PCR analyses reveal that microRNA-17-92-containing eccDNAs, along with the expression of their corresponding microRNAs, are elevated in HCC tumors and associated with poor outcomes and the age of HCC patients. More intriguingly, exogenous expression of artificial DNA circles harboring the miR-17-92 cluster, which are synthesized by the ligase-assisted minicircle accumulation method, can significantly accelerate HCC cell proliferation and migration.These findings delineate the genome-wide eccDNAs profiling of HCC and highlight the functional significance of microRNA-containing eccDNAs in tumorigenesis, providing insight into HCC pathogenesis and cancer therapy, as well as eccDNA and microRNA biology.Copyright © 2023 American Association for the Study of Liver Diseases.