MicroRNAs (miRNAs) are key players in human hepatocellular carcinoma (HCC) tumorigenesis. Therefore, small molecules targeting components of miRNA biogenesis may provide new therapeutic means for HCC treatment. By a high-throughput screening and structural simplification, we identified a small molecule, CIB-3b, which suppresses the growth and metastasis of HCC in vitro and in vivo by modulating expression profiles of miRNAome and proteome in HCC cells. Mechanistically, CIB-3b physically binds to transactivation response (TAR) RNA-binding protein 2 (TRBP) and disrupts the TRBP-Dicer interaction, thereby altering the activity of Dicer and mature miRNA production. Structure-activity relationship study via the synthesis of 45 CIB-3b derivatives showed that some compounds exhibited a similar inhibitory effect on miRNA biogenesis to CIB-3b. These results support TRBP as a potential therapeutic target in HCC and warrant further development of CIB-3b along with its analogues as a novel therapeutic strategy for the treatment of HCC.