Recent studies have revealed that repressor/activator protein (Rap1) not only protects telomeres from sister chromatid exchange, but also functions in genomewide transcriptional regulation. Knockdown of Rap1 sensitizes breast cancer cells to adriamycin-induced apoptosis. However, little is known about the role of Rap1 in the progression of hepatocellular carcinoma (HCC). The present study aimed to investigate the functions of Rap1 in HCC progression and to determine whether targeting the Rap1 signaling pathway may be of therapeutic value against HCC. We found knockdown of Rap1 by microRNA (miRNA) interference enhanced significantly apoptosis and 5-fluorouracil (5-FU) chemosensitivity in HepG2 cell line. Rap1 miRNA downregulated nuclear factor-κB p65 (NF-κB p65) expression, and upregulated inhibitor of NF-κB (IκB) expression. In vivo, Rap1 miRNA combined with 5-FU treatment led to a significant reduction of tumor growth as compared with 5-FU alone. The results indicate that Rap1 miRNA can effectively enhance sensitivity of HepG2 cell line to 5-FU chemotherapy in vitro and in vivo.